OP0094 Slit2/robo4 axis may contribute to angiogenesis disturbance in systemic sclerosis (SSC)
Authors: E. Romano, M. Manetti, I. Rosa, B.S. Fioretto, L. Ibba-Manneschi, M. Matucci-Cerinic, S. Guiducci
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> 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 <i>in vitro</i> and neovascularization <i>in vivo</i>. <h3>Objectives</h3> To evaluate the possible involvement of the Slit/Robo axis in SSc defective angiogenesis. <h3>Methods</h3> 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. <h3>Results</h3> 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&lt;0.0005), while Slit2 concentrations were similar in VEDOSS with normal NVC and controls. In SSc, Slit2 and Robo4 expression was higher in clinically affected skin and cultured MVECs in respect to controls. No difference was found in Robo1 expression. Cell viability, wound healing capacity and capillary-like tube formation in H-MVECs were all significantly reduced after challenge with rh Slit2 or SSc sera. These inhibitory effects were significantly attenuated when SSc sera were preincubated with anti-Slit2 blocking antibody. Cell viability, wound healing capacity and <i>in vitro</i> angiogenesis were severely compromised in SSc-MVECs and could be significantly ameliorated by Slit2 neutralisation or Robo4 gene silencing. <h3>Conclusions</h3> 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. <h3>Disclosure of Interest</h3> None declared