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Background: A consensus on digital ulcer (DU) definition in systemic sclerosis (SSc) has been recently reached (Suliman et al., J Scleroderma Relat Disord 2:115-20, 2017), while for their evaluation, classification and categorisation, it is still missing. The aims of this study were to identify a set of essential items for digital ulcer (DU) evaluation, to assess if the existing DU classification was useful and feasible in clinical practice and to investigate if the new categorisation was preferred to the simple distinction of DU in recurrent and not recurrent, in patients with systemic sclerosis (SSc)., Methods: DeSScipher is the largest European multicentre study on SSc. It consists of five observational trials (OTs), and one of them, OT1, is focused on DU management. The DeSScipher OT1 items on DU that reached ≥ 60% of completion rate were administered to EUSTAR (European Scleroderma Trials and Research group) centres via online survey. Questions about feasibility and usefulness of the existing DU classification (DU due to digital pitting scars, to loss of tissue, derived from calcinosis and gangrene) and newly proposed categorisation (episodic, recurrent and chronic) were also asked., Results: A total of 84/148 (56.8%) EUSTAR centres completed the questionnaire. DeSScipher items scored by ≥ 70% of the participants as essential and feasible for DU evaluation were the number of DU defined as a loss of tissue (level of agreement 92%), recurrent DU (84%) and number of new DU (74%). For 65% of the centres, the proposed classification of DU was considered useful and feasible in clinical practice. Moreover, 80% of the centres preferred the categorisation of DU in episodic, recurrent and chronic to simple distinction in recurrent/not recurrent DU., Conclusions: For clinical practice, EUSTAR centres identified only three essential items for DU evaluation and considered the proposed classification and categorisation as useful and feasible. The set of items needs to be validated while further implementation of DU classification and categorisation is warranted., Trial Registration: Observational trial on DU (OT1) is one of the five trials of the DeSScipher project (ClinicalTrials.gov; OT1 Identifier: NCT01836263 , posted on April 19, 2013).

Background: Systemic sclerosis (SSc) is characterized by endothelial cell (EC) apoptosis, impaired angiogenesis and peripheral microvasculopathy. Soluble α-Klotho (sKl) is a pleiotropic molecule with multiple effects on ECs, including antioxidant and vasculoprotective activities. On the EC surface, sKl interacts with vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) and transient receptor potential canonical-1 (TRPC-1) cation channel to control EC homeostasis. Here, we investigated whether sKl might act as a protective factor to improve angiogenesis in dermal microvascular endothelial cells (MVECs) from SSc patients (SSc-MVECs)., Methods: Wound healing assay was performed on healthy dermal MVECs (H-MVECs) challenged with sera from healthy controls or SSc patients with or without the addition of sKl. Capillary morphogenesis on Matrigel was assessed in H-MVECs and SSc-MVECs at basal conditions and treated with sKl, as well as in H-MVECs challenged with healthy or SSc sera in presence or absence of sKl. The expression of α-Klotho, VEGF 165 b, VEGFR-2, TRPC-1, Ki67 and active caspase-3 in H-MVECs and SSc-MVECs was investigated by western blotting. Immunostaining for α-Klotho was performed in H-MVECs and SSc-MVECs, and in healthy and SSc skin sections., Results: Treatment with sKl effectively counteracted the inihibitory effects of SSc sera on wound healing ability and angiogenic performance of H-MVECs. The addition of sKl significantly improved angiogenesis and maintained over time capillary-like tube formation in vitro by SSc-MVECs. Stimulation of SSc-MVECs with sKl resulted in the upregulation of the proliferation marker Ki67 in parallel with the downregulation of proapoptotic active caspase-3. The expression of α-Klotho was significantly lower in SSc-MVECs than in H-MVECs. The expression of TRPC-1 was also significantly decreased, while that of VEGFR-2 and VEGF 165 b was significantly increased, in SSc-MVECs compared with H-MVECs. Challenge with sKl either significantly increased TRPC-1 or decreased VEGF 165 b in SSc-MVECs. Ex vivo analyses revealed that α-Klotho immunostaining was almost absent in the dermal microvascular network of SSc skin compared with control skin., Conclusions: Our findings provide the first evidence that α-Klotho is significantly decreased in the microvasculature in SSc skin and that sKl administration may effectively improve SSc-MVEC functions in vitro by acting as a powerful proangiogenic factor.

Introduction: The vascular and nervous systems have several anatomic and molecular mechanism similarities. Emerging evidence suggests that proteins involved in transmitting axonal guidance cues, including members of class III semaphorin (Sema3) family, play a critical role in blood vessel guidance during physiological and pathological vascular development. Sema3E is a natural antiangiogenic molecule that causes filopodial retraction in endothelial cells, inhibiting cell adhesion by disrupting integrin-mediated adhesive structures. The aim of the present study was to investigate whether in systemic sclerosis (SSc) Plexin-D1/Sema3E axis could be involved in the dysregulation of vascular tone control and angiogenesis., Methods: Sema3E levels were measured by quantitative colorimetric sandwich ELISA in serum samples from 48 SSc patients, 45 subjects with primary Raynaud's phenomenon (pRP) and 48 age-matched and sex-matched healthy controls. Immunofluorescence staining on skin sections from 14 SSc patients and 12 healthy subjects was performed to evaluate Sema3E and Plexin-D1 expression. Western blotting was used to assess Plexin-D1/Sema3E axis in human SSc and healthy dermal microvascular endothelial cells (SSc-MVECs and H-MVECs, respectively) at basal condition and after stimulation with recombinant human vascular endothelial growth factor (VEGF), SSc and healthy sera. Capillary morphogenesis on Matrigel was performed on H-MVECs treated with healthy, pRP or SSc sera in the presence of Sema3E and Plexin-D1 soluble peptides., Results: Serum Sema3E levels were significantly higher both in pRP subjects and SSc patients than in controls. In SSc, Sema3E levels were significantly increased in patients with early nailfold videocapillaroscopy (NVC) pattern compared to active/late patterns and pRP, and in patients without digital ulcers versus those with ulcers. In SSc skin, Sema3E expression was strongly increased in the microvascular endothelium. Cultured SSc-MVECs showed higher levels of phosphorylated Plexin-D1 and Sema3E expression than H-MVECs, and stimulation with SSc sera increased phosphorylated Plexin-D1 and Sema3E in H-MVECs. The addition of Sema3E-binding Plexin-D1 soluble peptide significantly attenuated the antiangiogenic effect of SSc sera on H-MVECs., Conclusions: Our findings suggest that Plexin-D1/Sema3E axis is triggered in SSc endothelium and may have a role in the dysregulation of angiogenesis and vascular tone control by inducing neuro-vascular mechanism alterations clinically evident in particular in the early disease phases.

Introduction: Microvascular damage and defective angiogenesis and vasculogenesis have a major role in the pathogenesis of systemic sclerosis (SSc). Epidermal growth factor-like domain 7 (EGFL7) is a proangiogenic molecule which is predominantly expressed and secreted by endothelial cells and their progenitors and controls vascular development and integrity. In this study, we investigated the possible involvement of EGFL7 in SSc., Methods: Serum EGFL7 levels from 60 patients with SSc and 35 age- and sex-matched healthy controls were examined by colorimetric sandwich enzyme-linked immunosorbent assay. The expression of EGFL7 in forearm skin biopsies (n = 16 SSc, n = 10 controls), cultured dermal microvascular endothelial cells (MVECs) (n = 3 SSc, n = 3 controls) and late-outgrowth peripheral blood endothelial progenitor cell (EPC)-derived endothelial cells (n = 15 SSc, n = 8 controls) was investigated by immunofluorescence and Western blotting., Results: Serum EGFL7 levels were detectable in 68.6% of healthy controls and 45% of SSc cases (P < 0.05). Circulating levels of EGFL7 were significantly decreased in SSc patients compared with healthy controls (P = 0.01). Serum levels of EGFL7 were significantly lower in both limited cutaneous SSc and diffuse cutaneous SSc patients than in controls (P = 0.02 and P = 0.04, respectively). In SSc, decreased serum EGFL7 levels were significantly correlated with the severity of nailfold capillary abnormalities. Patients with the most severe capillary changes and digital ulcers had serum EGFL7 levels significantly lower than healthy controls, while the EGFL7 levels did not differ significantly between controls and SSc patients with less capillary damage and lack of digital ulcers. Endothelial EGFL7 expression was strongly downregulated or even almost completely undetectable in SSc-affected dermis compared with controls (P < 0.001). In cultured SSc dermal MVECs and late-outgrowth peripheral blood EPC-derived endothelial cells, EGFL7 was significantly downregulated compared with cells obtained from healthy subjects (P < 0.01 and P < 0.001, respectively)., Conclusions: Our findings suggest that the loss of EGFL7 expression in endothelial cells and their progenitors might play a role in the development and progression of peripheral microvascular damage and the defective vascular repair process characteristic of SSc.