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Systemic sclerosis (SSc, scleroderma) is a complex connective tissue disorder characterized by multisystem clinical manifestations resulting from immune dysregulation/autoimmunity, vasculopathy, and, most notably, progressive fibrosis of the skin and internal organs. In recent years, it has been observed that the main drivers of SSc-related tissue fibrosis are myofibroblasts, a type of mesenchymal cells with both the extracellular matrix-synthesizing features of fibroblasts and the cytoskeletal characteristics of contractile smooth muscle cells. The accumulation and persistent activation of pro-fibrotic myofibroblasts during SSc development and progression result in elevated mechanical stress and reduced matrix plasticity within the affected tissues and may be ascribed to a reduced susceptibility of these cells to pro-apoptotic stimuli, as well as their increased formation from tissue-resident fibroblasts or transition from different cell types. Given the crucial role of myofibroblasts in SSc pathogenesis, finding the way to inhibit myofibroblast differentiation and accumulation by targeting their formation, function, and survival may represent an effective approach to hamper the fibrotic process or even halt or reverse established fibrosis. In this review, we discuss the role of myofibroblasts in SSc-related fibrosis, with a special focus on their cellular origin and the signaling pathways implicated in their formation and persistent activation. Furthermore, we provide an overview of potential therapeutic strategies targeting myofibroblasts that may be able to counteract fibrosis in this pathological condition.

Objectives We characterized the microbiota in systemic sclerosis (SSc), focusing on the skin-oral-gut axis and serum and faecal free fatty acid (FFA) profile. Methods Twenty-five SSc patients with ACA or anti-Scl70 autoantibodies were enrolled. The microbiota of faecal, saliva and superficial epidermal samples was assessed through next generation-sequencing analysis. Gas chromatography-mass spectroscopy was used to quantity faecal and serum FFAs. Gastrointestinal symptoms were investigated with UCLA GIT-2.0-questionnaire. Results The ACA+ and anti-Scl70+ groups displayed different cutaneous and faecal microbiota profiles. The classes of cutaneous Sphingobacteria and Alphaproteobacteria, the faecal phylum of Lentisphaerae, the classes of Lentisphaeria and Opitutae, and the genus of NA-Acidaminococcaceae were significantly higher in faecal samples of the ACA+, compared with anti-Scl70+ patients. The cutaneous Sphingobacteria and the faecal Lentisphaerae were significantly correlated (rho = 0.42; p= 0.03). A significant increase in faecal propionic acid was observed in ACA+ patients. Moreover, all faecal medium-chain FFAs and hexanoic acids were significantly higher in ACA+ compared with anti-Scl70+ group (p< 0.05 and p< 0.001, respectively). In the ACA+ group, the analysis of serum FFA levels showed an increasing trend in valeric acid. Conclusions Different microbiota signatures and FFA profiles were found in the two groups of patients. Despite being in different body districts, the cutaneous Sphingobacteria and faecal Lentisphaerae appear interdependent.

Corneal transparency, necessary for vision and depending on the high organization of stromal extracellular matrix, is maintained by keratocytes. Severe or continuous corneal injuries determine exaggerated healing responses resulting in the formation of irreversible fibrotic scars and vision impairment. Soluble guanylate cyclase (sGC) stimulation demonstrated antifibrotic effects in both experimental fibrosis and human lung and skin fibroblasts. Here, we assessed whether sGC stimulation with BAY 41-2272 could attenuate transforming growth factor β1 (TGFβ1)-induced myofibroblast differentiation of human corneal keratocytes. Cells were challenged with TGFβ1, with/without BAY 41-2272 preincubation, and subsequently assessed for viability, proliferation, migration, chemoinvasion, as well for the expression of myofibroblast/fibroblast activation markers and contractile abilities. Treatment with BAY 41-2272 did not affect keratocyte viability, while preincubation of cells with the sGC stimulator was able to inhibit TGFβ1-induced proliferation, wound healing capacity, and invasiveness. BAY 41-2272 was also able to attenuate TGFβ1-induced myofibroblast-like profibrotic phenotype of keratocytes, as demonstrated by the significant decrease in ACTA2, COL1A1, COL1A2, FN1 and PDPN gene expression, as well as in α-smooth muscle actin, α-1 chain of type I collagen, podoplanin, vimentin and N-cadherin protein expression. Finally, BAY 41-2272 significantly counteracted the TGFβ1-induced myofibroblast-like ability of keratocytes to contract collagen gels, reduced phosphorylated Smad3 protein levels, and attenuated gene expression of proinflammatory cytokines. Collectively, our data show for the first time that BAY 41-2272 is effective in counteracting keratocyte-to-myofibroblast transition, thus providing the rationale for the development of sGC stimulators as novel promising modulators of corneal scarring and fibrosis.

Adipose-derived stem cells (ADSCs) residing in the stromal vascular fraction (SVF) of white adipose tissue are recently emerging as an alternative tool for stem cell-based therapy in systemic sclerosis (SSc), a complex connective tissue disorder affecting the skin and internal organs with fibrotic and vascular lesions. Several preclinical and clinical studies have reported promising therapeutic effects of fat grafting and autologous SVF/ADSC-based local treatment for facial and hand cutaneous manifestations of SSc patients. However, currently available data indicate that ADSCs may represent a double-edged sword in SSc, as they may exhibit a pro-fibrotic and anti-adipogenic phenotype, possibly behaving as an additional pathogenic source of pro-fibrotic myofibroblasts through the adipocyte-to-myofibroblast transition process. Thus, in the perspective of a larger employ of SSc-ADSCs for further therapeutic applications, it is important to definitely unravel whether these cells present a comparable phenotype and similar immunosuppressive, anti-inflammatory, anti-fibrotic and pro-angiogenic properties in respect to healthy ADSCs. In light of the dual role that ADSCs seem to play in SSc, this review will provide a summary of the most recent insights into the preclinical and clinical studies employing SVF and ADSCs for the treatment of the disease and, at the same time, will focus on the main findings highlighting the possible involvement of these stem cells in SSc-related fibrosis pathogenesis.

Objectives In SSc, angiogenesis impairment advances in parallel with the development of fibrosis orchestrated by myofibroblasts originating from different sources, including endothelial-to-mesenchymal transition (EndoMT). Soluble guanylate cyclase (sGC) stimulation has shown antifibrotic effects in SSc skin fibroblasts and mouse models. Here, we investigated the effects of pharmacological sGC stimulation on impaired angiogenesis and myofibroblast-like features of SSc dermal microvascular endothelial cells (SSc-MVECs). Methods To determine whether sGC stimulation affected cell viability/proliferation, SSc-MVECs and healthy dermal MVECs (H-MVECs) were challenged with the sGC stimulator (sGCS) MK-2947 and assayed by annexin V/propidium iodide flow cytometry and the water-soluble tetrazolium salt (WST-1) assay. To study angiogenesis and EndoMT, MK-2947-treated SSc-MVECs were subjected to wound healing and capillary morphogenesis assays and analysed for the expression of endothelial/myofibroblast markers and contractile ability. Results MK-2947 treatment did not affect H-MVEC viability/proliferation, while it significantly increased SSc-MVEC proliferation, wound healing capability and angiogenic performance. After MK-2947 treatment, SSc-MVECs exhibited significantly increased proangiogenic MMP9 and decreased antiangiogenic MMP12 and PTX3 gene expression. A significant increase in the expression of CD31 and vascular endothelial cadherin paralleled by a decrease in α-smooth muscle actin, S100A4, type I collagen and Snail1 mesenchymal markers was also found in MK-2947-treated SSc-MVECs. Furthermore, stimulation of sGC with MK-2947 significantly counteracted the intrinsic ability of SSc-MVECs to contract collagen gels and reduced phosphorylated-extracellular signal-regulated kinases 1 and 2 protein levels. Conclusion These findings demonstrate for the first time that pharmacological sGC stimulation effectively ameliorates the angiogenic performance and blunts the myofibroblast-like profibrotic phenotype of SSc-MVECs, thus providing new evidence for repurposing sGCSs for SSc.

Systemic sclerosis (SSc, scleroderma) is a multifaceted rare connective tissue disease whose pathogenesis is dominated by immune dysregulation, small vessel vasculopathy, impaired angiogenesis, and both cutaneous and visceral fibrosis. Microvascular impairment represents the initial event of the disease, preceding fibrosis by months or years and accounting for the main disabling and/or life-threatening clinical manifestations, including telangiectasias, pitting scars, periungual microvascular abnormalities (e.g., giant capillaries, hemorrhages, avascular areas, ramified/bushy capillaries) clinically detectable by nailfold videocapillaroscopy, ischemic digital ulcers, pulmonary arterial hypertension, and scleroderma renal crisis. Despite a variety of available treatment options, treatment of SSc-related vascular disease remains problematic, even considering SSc etherogenity and the quite narrow therapeutic window. In this context, plenty of studies have highlighted the great usefulness in clinical practice of vascular biomarkers allowing clinicians to assess the evolution of the pathological process affecting the vessels, as well as to predict the prognosis and the response to therapy. The current narrative review provides an up-to-date overview of the main candidate vascular biomarkers that have been proposed for SSc, focusing on their main reported associations with characteristic clinical vascular features of the disease.

Considerable evidence accumulated over the past decade supports that telocytes (TCs)/CD34+ stromal cells represent an exclusive type of interstitial cells identifiable by transmission electron microscopy (TEM) or immunohistochemistry in various organs of the human body, including the skin. By means of their characteristic cellular extensions (telopodes), dermal TCs are arranged in networks intermingled with a multitude of neighboring cells and, hence, they are thought to contribute to skin homeostasis through both intercellular contacts and releasing extracellular vesicles. In this context, fibrotic skin lesions from patients with systemic sclerosis (SSc, scleroderma) appear to be characterized by a disruption of the dermal network of TCs, which has been ascribed to either cell degenerative processes or possible transformation into profibrotic myofibroblasts. In the present study, we utilized the well-established mouse model of bleomycin-induced scleroderma to gain further insights into the TC alterations found in cutaneous fibrosis. CD34 immunofluorescence revealed a severe impairment in the dermal network of TCs/CD34+ stromal cells in bleomycin-treated mice. CD31/CD34 double immunofluorescence confirmed that CD31−/CD34+ TC counts were greatly reduced in the skin of bleomycin-treated mice compared with control mice. Ultrastructural signs of TC injury were detected in the skin of bleomycin-treated mice by TEM. The analyses of skin samples from mice treated with bleomycin for different times by either TEM or double immunostaining and immunoblotting for the CD34/α-SMA antigens collectively suggested that, although a few TCs may transition to α-SMA+ myofibroblasts in the early disease stage, most of these cells rather undergo degeneration, and then are lost. Taken together, our data demonstrate that TC changes in the skin of bleomycin-treated mice mimic very closely those observed in human SSc skin, which makes this experimental model a suitable tool to (i) unravel the pathological mechanisms underlying TC damage and (ii) clarify the possible contribution of the TC loss to the development/progression of dermal fibrosis. In perspective, these findings may have important implications in the field of skin regenerative medicine.

In systemic sclerosis (SSc), abnormalities in microvessel morphology occur early and evolve into a distinctive vasculopathy that relentlessly advances in parallel with the development of tissue fibrosis orchestrated by myofibroblasts in nearly all affected organs. Our knowledge of the cellular and molecular mechanisms underlying such a unique relationship between SSc-related vasculopathy and fibrosis has profoundly changed over the last few years. Indeed, increasing evidence has suggested that endothelial-to-mesenchymal transition (EndoMT), a process in which profibrotic myofibroblasts originate from endothelial cells, may take center stage in SSc pathogenesis. While in arterioles and small arteries EndoMT may lead to the accumulation of myofibroblasts within the vessel wall and development of fibroproliferative vascular lesions, in capillary vessels it may instead result in vascular destruction and formation of myofibroblasts that migrate into the perivascular space with consequent tissue fibrosis and microvessel rarefaction, which are hallmarks of SSc. Besides endothelial cells, other vascular wall-resident cells, such as pericytes and vascular smooth muscle cells, may acquire a myofibroblast-like synthetic phenotype contributing to both SSc-related vascular dysfunction and fibrosis. A deeper understanding of the mechanisms underlying the differentiation of myofibroblasts inside the vessel wall provides the rationale for novel targeted therapeutic strategies for the treatment of SSc.

Telocytes (TCs), commonly referred to as TCs/CD34+ stromal cells, are a peculiar type of interstitial cells with distinctive morphologic traits that are supposed to exert several biological functions, including tissue homeostasis regulation, cell-to-cell signaling, immune surveillance, and reparative/regenerative effects. At present, the majority of studies investigating these cells are mainly descriptive and focus only on their morphology, with a consequent paucity of functional data. To gain relevant insight into the possible functions of TCs, in vitro analyses are clearly required, but currently, the protocols for TC isolation are only at the early stages and not fully standardized. In the present in vitro study, we describe a novel methodology for the purification of human primary skin TCs through a two-step immunomagnetic microbead-based cell separation (i.e., negative selection for CD31 followed by positive selection for CD34) capable of discriminating these cells from other connective tissue-resident cells on the basis of their different immunophenotypic features. Our experiments clearly demonstrated that the proposed method allows a selective purification of cells exhibiting the peculiar TC morphology. Isolated TCs displayed very long cytoplasmic extensions with a moniliform silhouette (telopodes) and presented an immunophenotypic profile (CD31&minus, /CD34+/PDGFR&alpha, +/vimentin+) that unequivocally differentiates them from endothelial cells (CD31+/CD34+/PDGFR&alpha, &minus, /vimentin+) and fibroblasts (CD31&minus, /CD34&minus, /PDGFR&alpha, +/vimentin+). This novel methodology for the isolation of TCs lays the groundwork for further research aimed at elucidating their functional properties and possible translational applications, especially in the field of regenerative medicine.

Systemic sclerosis (SSc) is a life-threatening connective tissue disorder of unknown etiology characterized by widespread vascular injury and dysfunction, impaired angiogenesis, immune dysregulation and progressive fibrosis of the skin and internal organs. Over the past few years, a new trend of investigations is increasingly reporting aberrant epigenetic modifications in genes related to the pathogenesis of SSc, suggesting that, besides genetics, epigenetics may play a pivotal role in disease development and clinical manifestations. Like many other autoimmune diseases, SSc presents a striking female predominance, and even if the reason for this gender imbalance has yet to be completely understood, it appears that the X chromosome, which contains many gender and immune-related genes, could play a role in such gender-biased prevalence. Besides a short summary of the genetic background of SSc, in this review we provide a comprehensive overview of the most recent insights into the epigenetic modifications which underlie the pathophysiology of SSc. A particular focus is given to genetic variations in genes located on the X chromosome as well as to the main X-linked epigenetic modifications that can influence SSc susceptibility and clinical phenotype. On the basis of the most recent advances, there is realistic hope that integrating epigenetic data with genomic, transcriptomic, proteomic and metabolomic analyses may provide in the future a better picture of their functional implications in SSc, paving the right way for a better understanding of disease pathogenesis and the development of innovative therapeutic approaches.

Systemic sclerosis (SSc, scleroderma) is a severe autoimmune connective tissue disease characterized by widespread peripheral microvasculopathy, and progressive cutaneous and visceral fibrosis, leading to significant organ dysfunction. Sirtuins (SIRTs) are a family of NAD-dependent protein deacetylases with pleiotropic effects on a variety of biological processes, including metabolism, cell survival, and aging. In the last decades, increasing studies have explored the contribution of SIRTs to the pathogenesis of SSc, highlighting a significant antifibrotic effect of both SIRT1 and SIRT3. On these bases, the aim of this study was to measure circulating SIRT1 and SIRT3 levels by enzyme-linked immune-sorbent assay in a well-characterized cohort of SSc patients (n = 80) and healthy controls (n = 71), focusing on their possible association with disease clinical features, and their potential as biomarkers reflecting SSc activity and severity. Significantly decreased serum levels of both SIRT1 and SIRT3 were found in SSc patients compared to controls. In SSc, the reduction in circulating SIRT1 and SIRT3 associated with a greater extent of cutaneous fibrosis, presence of interstitial lung disease, and worse pulmonary function. Serum SIRT1 and SIRT3 decrease also correlated with the severity of nailfold microvascular damage, with SIRT3 levels being additionally related to the occurrence of digital ulcers. The levels of these two proteins showed a direct correlation with one another in the circulation of SSc patients. Of the two SIRTs, serum SIRT3 was found to better reflect disease activity and severity in a logistic regression analysis model. Our findings suggest that serum SIRT1 and SIRT3 may represent novel potential biomarkers of increased risk for a more severe, life-threatening SSc disease course.

Systemic sclerosis (SSc) is a multifaceted connective tissue disease characterized by widespread vasculopathy and autoimmune reactions that evolve into progressive interstitial, perivascular, and vessel wall fibrosis that affects the skin and multiple internal organs. Such an uncontrolled fibrotic process gradually disrupts the physiologic architecture of the affected tissues and frequently leads to significant organ dysfunction, thus representing a major cause of death in SSc patients. The main fibrosis orchestrators in SSc are represented by chronically activated myofibroblasts, a peculiar population of mesenchymal cells combining the extracellular matrix-synthesizing features of fibroblasts with cytoskeletal characteristics of contractile smooth muscle cells. Multiple lines of evidence support the notion that profibrotic myofibroblasts may derive not only from the activation of tissue resident fibroblasts but also from a variety of additional cell types, including pericytes, epithelial cells, vascular endothelial cells and preadipocytes/adipocytes. Here we overview an emerging picture that espouses that several cell transitional processes may be novel essential contributors to the pool of profibrotic myofibroblasts in SSc, potentially representing new suitable targets for therapeutic purposes. An in-depth dissection of the multiple origins of myofibroblasts and the underlying molecular mechanisms may be crucial in the process of deciphering the cellular bases of fibrosis persistence and refractoriness to the treatment and, therefore, may help in developing more effective and personalized therapeutic opportunities for SSc patients.

Background:In systemic sclerosis (SSc), alternative splicing of the last exon (exon 8) of vascular endothelial growth factor (VEGF)-A pre-mRNA is a key element in the switch from proangiogenic to antiangiogenic VEGF-A isoforms. The mRNA-binding protein serine/arginine protein 55 (SRp55, also known as SFRS6) is a key regulatory splicing factor that promotes distal splice-site selection in the exon 8 region of VEGF-A pre-mRNA and subsequent upregulation of the exon 8b-containing VEGF165b antiangiogenic isoform. Overexpression of both VEGF165b and SRp55 has been implicated in SSc-related angiogenesis impairment and peripheral vascular damage. Moreover, differential splicing of the VEGF-A gene has been shown to be critical for development of pulmonary fibrosis. Of note, previous studies reported the lack of sequence variations in the VEGF-A alternatively spliced region, while a single nucleotide polymorphism (SNP) in theSRp55gene (rs2235611) has been associated with susceptibility to disturbed ocular angiogenesis in proliferative diabetic retinopathy.Objectives:This case-control pilot study examined the possible implication ofSRp55rs2235611 SNP in the genetic predisposition to SSc susceptibility and clinical phenotype.Methods:A total population of 872 white Italian individuals (414 SSc patients, 458 controls) was studied. All patients were classified as limited and diffuse cutaneous SSc (lcSSc and dcSSc, respectively) and were clinically evaluated for the presence of autoantibodies (anticentromere, anti-Scl70 antibodies), pulmonary fibrosis and digital ulcers. TheSRp55rs2235611 SNP was genotyped by TaqMan Real-Time PCR.Results:SRp55rs2235611 genotype distribution and allele frequency were similar in SSc and healthy controls, though a trend toward significance was observed for genotype distribution (p=0.07). TheSRp55rs2235611 AA genotype significantly influenced the predisposition to SSc (OR 2.55, 95% CI 1.11 to 5.57, p = 0.03), and to both lcSSc (OR 2.80, 95% CI 1.16 to 6.84, p = 0.02) and dcSSc (OR 3.42, 95% CI 1.20 to 9.72, p = 0.02) subtypes. TheSRp55rs2235611 A minor allele and AA genotype showed a significant risk association with susceptibility to SSc-related pulmonary fibrosis (A allele: OR 1.39, 95% CI 1.00 to 1.93, p = 0.046; AA genotype: OR 3.95, 95% CI 1.48 to 10.54, p = 0.006). A trend toward an association between the AA genotype and anti-Scl70 antibody-positive SSc was also found (OR 2.82, 95% CI 0.95 to 8.37, p = 0.06). Both rs2235611 A allele and AA genotype were significantly associated with the SSc subset without digital ulcers (A allele: OR 1.33, 95% CI 1.01 to 1.75, p = 0.04; AA genotype: OR 3.26, 95% CI 1.32 to 8.03, p = 0.01).Conclusion:TheSRp55rs2235611 polymorphism is associated with susceptibility to SSc and, in particular, with SSc-related pulmonary fibrosis and peripheral vascular phenotype, consistent with a role of VEGF-A pre-mRNA alternative splicing in the development of pulmonary fibrosis and impairment of angiogenesis. Further replication studies are warranted to confirm our findings in independent SSc cohorts.Disclosure of Interests:Eloisa Romano: None declared, Mirko Manetti: None declared, Joanna Kosalka-Wegiel: None declared, Bianca Saveria Fioretto: None declared, Irene Rosa: None declared, Elena Sticchi: None declared, Serena Guiducci: None declared, Silvia Bellando-Randone: None declared, Lidia Ibba-Manneschi: None declared, Marco Matucci-Cerinic Grant/research support from: Actelion, MSD, Bristol-Myers Squibb, Speakers bureau: Acetelion, Lilly, Boehringer Ingelheim

Systemic sclerosis (SSc; scleroderma) is characterized by life-threatening progressive multiorgan fibrosis orchestrated by profibrotic myofibroblasts originating from different sources. Because recent data demonstrated that the majority of myofibroblasts in a murine scleroderma model arise from adipocytic progenitors through the adipocyte-myofibroblast transition process, we sought to determine whether the SSc microenvironment may affect the differentiation potential of adipose-derived stem cells (ADSC). Normal human ADSC from three donors were treated with serum from SSc patients (n = 6), serum from healthy individuals (n = 6), or recombinant human transforming growth factor-β1 (TGFβ1) as positive control of myofibroblastic phenotype induction. ADSC were subjected to in vitro adipogenic differentiation for up to 21 days in the presence of different stimuli followed by lipid content quantification. In selected experiments, adipocytic and mesenchymal/myofibroblast marker gene and protein expression levels were assessed by Real-Time PCR, immunoblotting and immunofluorescence after administration of different stimuli for 72 and 96 h, respectively. Cell contractile phenotype was assayed by collagen gel contraction assay. Likewise stimulation with TGFβ1, SSc serum was able to significantly inhibit the adipocyte differentiation of ADSC as testified by a strong decrease in red-colored lipid droplets after 21 days of adipogenic induction. Treatment of ADSC either with SSc serum or TGFβ1 resulted in the acquisition of a myofibroblast-like phenotype characterized by a reduced expression of the adipocytic markers perilipin and adiponectin, a significant upregulation of the mesenchymal/myofibroblast markers α-SMA+ stress fibers, S100A4 and type I collagen, and an ability to effectively contract collagen gels. In SSc, the pathologic environment may favor the differentiation of ADSC into profibrotic and contractile myofibroblast-like cells. These findings strengthen the notion that the generation of myofibroblasts from ADSC may be relevant in SSc pathophysiology potentially representing a new target for the prevention/treatment of multiorgan fibrosis.

Systemic sclerosis (SSc) is a connective tissue disorder characterised by immune dysregulation, endothelial cell dysfunction followed by defective vascular repair and neovascularization and progressive tissue fibrosis of the skin and internal organs, whose pathophysiology remains to be fully elucidated. Perturbed neuroendothelial control mechanisms comprising either endothelial cell or peripheral nerve fiber impairment are supposed to play an important role in the onset of Raynaud's phenomenon and development of microvascular abnormalities which are the earliest events and key features of SSc. Such pathogenic neuroendothelial mechanisms may trigger both the early endothelial cell damage and the subsequent loss of peripheral microvascular integrity characterised by the lack of compensatory angiogenesis. Of note, the vascular and nervous systems have several anatomical similarities that extend to molecular level, and the molecular mechanisms of nerve regulation are shared by the vascular system. In this context, increasing evidence demonstrated that endothelial cells express receptors for axon guidance molecules, including Ephrin family receptor tyrosine kinases, Neuropilins, Plexins, Robos, and UNC5B that are able to respond to their soluble neuroendothelial trophic ligands, such as Semaphorins and Slits, to guide the sprouting of endothelial tip cells. Here, we first provide a historical view of neuroendothelial control mechanism alterations in the pathogenesis of SSc, and then discuss the emerging role of a class of molecules sharing neurogenic and angiogenic properties, such as members of Semaphorin/Plexin/Neuropilin and Slit/Roundabout families, in SSc-related peripheral microvasculopathy.

Background In SSc, vascular involvement is a primary event characterised by vascular tone dysfunction and microcirculatory abnormalities. Many classes of guidance molecules, such as members of the secreted glycoproteins Slits and their Roundabout (Robo) receptors, play critical roles in angiogenesis. Among these, Robo1 and Robo4 are expressed in endothelial cells. In particular, it has been demonstrated that the interaction of Slit2 with Robo1 promotes angiogenesis, while the Slit2/Robo4 axis inhibits VEGF-mediated endothelial cell migration and tube formation in vitro and neovascularization in vivo . Objectives To evaluate the possible involvement of the Slit/Robo axis in SSc defective angiogenesis. Methods Serum Slit2 levels were measured by ELISA in 78 SSc patients, 64 patients with a very early diagnosis of SSc (VEDOSS) and 74 age- and sex-matched healthy controls. Slit2, Robo1 and Robo4 protein expression was evaluated by immunofluorescence in skin biopsies from 15 SSc patients and 10 controls. Slit2 and Robo4 expression in dermal microvascular endothelial cells isolated from 5 SSc patients (SSc-MVECs) and 5 healthy controls (H-MVECs) was analysed by quantitative real-time PCR, Western blot and immunocytochemistry. Proliferation, wound healing and capillary-like tube formation were assessed in H-MVECs challenged with recombinant human (rh) Slit2 or SSc sera (n=6) in the presence/absence of an anti-Slit2 blocking antibody, as well as in SSc-MVECs treated with anti-Slit2 antibody or Robo4 siRNA. Results Circulating Slit2 levels were significantly increased in either SSc (median 11.12 ng/ml, IQR 8.02–16.25 ng/ml) or VEDOSS (median 11.27 ng/ml, IQR 8.46–18.60 ng/ml) compared with healthy controls (median 8.79 ng/ml, IQR 6.68–12.13 ng/ml) (p=0.002 and p=0.001, respectively). Serum Slit2 was elevated in SSc patients irrespective of the nailfold videocapillaroscopy (NVC) pattern or the presence/absence of digital ulcers. Interestingly, differences in serum Slit2 levels were found between VEDOSS patients with early/active NVC pattern and controls (p in vitro angiogenesis were severely compromised in SSc-MVECs and could be significantly ameliorated by Slit2 neutralisation or Robo4 gene silencing. Conclusions In SSc, increased circulating levels of Slit2 and activation of the Slit2/Robo4 antiangiogenic axis may contribute to peripheral microangiopathy since the very early phase of the disease. Disclosure of Interest None declared

ObjectiveIn systemic sclerosis (SSc), early microvascular injury is followed by impaired angiogenesis and peripheral capillary loss. Here, we investigated the possible contribution of the neurovascular guidance molecule Slit2 and its Roundabout (Robo) receptors to SSc-related endothelial cell dysfunction.MethodsCirculating Slit2 levels were measured in patients with SSc and healthy controls. Slit2, Robo1 and Robo4 expression was investigated in SSc and healthy skin biopsies and explanted dermal microvascular endothelial cells (MVECs). Slit2/Robo4 function in MVEC angiogenesis was studied by cell viability, wound healing and capillary-like tube formation assays.ResultsCirculating Slit2 was significantly increased in either SSc or patients with a very early diagnosis of SSc (VEDOSS) compared with controls. Interestingly, serum Slit2 levels were raised in patients with VEDOSS with nailfold videocapillaroscopy (NVC) abnormalities, while they were similar in VEDOSS with normal NVC and controls. In SSc, Slit2 and Robo4 expression was upregulated in clinically affected skin and explanted MVECs in respect to controls. The angiogenic performance of healthy MVECs was significantly reduced after challenge with recombinant human Slit2 or SSc sera. These inhibitory effects were significantly attenuated when SSc sera were preincubated with an anti-Slit2 blocking antibody. In vitro angiogenesis was severely compromised in SSc-MVECs and could be significantly ameliorated by Slit2 neutralisation or ROBO4 gene silencing. Slit2/Robo4 axis interfered with angiogenesis through the inhibition of Src kinase phosphorylation.ConclusionsIn SSc, increased circulating levels of Slit2 and activation of the Slit2/Robo4 antiangiogenic axis may contribute to peripheral microangiopathy since the very early phase of the disease.

Uncaria tomentosa (Willd.) DC. (Rubiacee), also known as uña de gato, is a plant that grows wild in the upper Amazon region of Peru and has been widely used in folk medicine to treat several health conditions including cancer. We have produced an aqueous extract from Uncaria tomentosa (UT-ex) and analyzed its effects on squamous carcinoma cells and immortalized HaCaT keratinocytes. Squamous cell carcinoma (SCC) is an uncontrolled growth of abnormal cells arising in the skin's squamous layer of epidermis. When detected at an early stage, SCCs are almost curable, however, if left untreated, they can penetrate the underlying tissue and become disfiguring. We have evaluated cell proliferation, apoptosis and the level of reactive oxygen species following UT-ex treatment. UT-ex affected cell cycle progression and reduced cell viability in a dose and time-dependent manner. From a mechanistic point of view, this delay in cell growth coincided with the increase of reactive oxygen species (ROS). Furthermore, PARP1 cleavage was associated to the reduction of Y-box binding protein 1 (YB-1) 36kDa, a nuclear prosurvival factor involved in DNA damage repair. These data indicate that UT-ex-induced cell death can be ascribed, at least in part, to its ability both to induce oxidative DNA damage and antagonize the mechanism of DNA repair relying upon YB-1 activity. They also show that non metastatic SCCs are more susceptible to UT-ex treatment than untransformed keratinocytes supporting the use of UT-ex for the treatment of precancerous and early forms of squamous cell carcinomas. Preliminary chemical investigation of UT-ex revealed the presence of hydrophilic low-medium molecular weight metabolites with anticancer potential towards squamous carcinoma cells.

In systemic sclerosis (SSc), the possible involvement of lymphatic microcirculation and lymphangiogenesis has traditionally been overshadowed by the greater emphasis placed on dysfunctional blood vascular system and angiogenesis. In the present in vitro study, we explore for the first time whether the SSc microenvironment may interfere with lymphangiogenesis, a complex, multi-step process in which lymphatic microvascular endothelial cells (LMVECs) sprout, migrate, and proliferate to generate new lymphatic capillaries. Normal human adult dermal LMVECs from three donors were treated with serum from SSc patients (n = 8), serum from healthy individuals (n = 8), or recombinant human vascular endothelial growth factor (VEGF)-C as a positive control for lymphangiogenesis. Cell proliferation, Boyden chamber Matrigel chemoinvasion, wound healing capacity, and lymphatic capillary morphogenesis on Geltrex were assayed. VEGF-C serum levels were measured by enzyme-linked immunosorbent assay. Gene and protein expression levels of the lymphangiogenic orchestrators VEGF receptor-3 (VEGFR-3)/Flt-4 and neuropilin-2 (NRP-2) were determined by real-time PCR and Western blotting, respectively. Conditioning with SSc serum significantly inhibited LMVEC proliferation, Matrigel invasion, and wound healing capacity with respect to healthy serum. The ability of LMVECs to form lymphatic tubes on Geltrex was also severely compromised in the presence of SSc serum. VEGF-C levels were comparable in SSc and healthy sera. Treatment with SSc serum resulted in a significant downregulation of both VEGFR-3/Flt-4 and NRP-2 mRNA and protein levels. In SSc, the pathologic environment severely hampers every lymphangiogenesis step, likely through the reduction of pro-lymphangiogenic VEGFR-3/NRP-2 co-receptor signaling. The impairment of the lymphangiogenic process opens a new scenario underlying SSc vascular pathophysiology, which is worth investigating further.