FRI0399 Rnai-based identification of novel drug targets to reduce endothelial to mesenchymal transition (ENDOMT) in human dermal microvascular endothelial cells: a target discovery approach for scleroderma
Authors: C. Corallo, J. Benschop, M. Grimbergen, J. Crawford, J. Meuldijk, R.A. Janssen, N.E. Vandeghinste, N. Giordano, M.A. Tessari
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Background</h3> Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterised by early vascular abnormalities and subsequent fibroblast activation and differentiation into myofibroblasts, leading to fibrosis.<sup>1,2</sup> Recently, endothelial to mesenchymal transition (EndoMT), a complex biological process in which endothelial cells lose their specific markers and acquire a mesenchymal or myofibroblastic phenotype, was reported in SSc.<sup>3</sup> <h3>Objectives</h3> We developed a high-content screening assay with the aim to identify novel proteins which, upon inhibition, will reduce EndoMT in SSc. <h3>Methods</h3> Human dermal microvascular endothelial cells (HMVECs) were seeded in 384 well plates and transfected with a RNAi-based library targeting 866 genes. Cells were then triggered with either disease-related cytokines, such as transforming growth factor beta (TGF-β), or with serum from very early diagnosed, limited cutaneous and diffuse cutaneous SSc patients. Serum derived from healthy donors was used as a control. After 72 hours of triggering, changes in expression level of endothelial and mesenchymal markers were quantified by immunocytochemistry and high content imaging. <h3>Results</h3> We developed a high-throughput EndoMT assay that allows for monitoring of changes in endothelial and mesenchymal markers upon triggering with disease-related cytokines and serum derived from SSc patients. We used this assay to screen an RNAi-based library in HMVECs and identify more than 100 targets able to reduce EndoMT triggered by either TGF-β or SSc patient serum. Identified hits subsequently will be extensively validated in secondary biological assays, and the validated targets will represent excellent candidate drug targets. <h3>Conclusions</h3> This program constitutes a critical path of experiments that will enable the selection of only those targets which meet pre-determined target acceptance criteria. We have chosen to identify targets in a human primary cell system and employ patient's serum as trigger of EndoMT to be able to closely investigate the cellular processes promoting EndoMT in SSc pathogenesis. By using this high-throughput screening platform, we aim to to find disease-modifying targets that will be used as an entry point for small molecule drug discovery. <h3>References</h3> [1] Denton CP. Advances in pathogenesis and treatment of systemic sclerosis.Clin Med (Lond) 2016;16:55–60. [2] Asano Y. Systemic Sclerosis. J Dermatol2017. doi:10.1111/1346-8138.14153 [3] Manetti M, Romano E, Rosa I, Guiducci S, Bellando-Randone S, De Paulis A, Ibba-Manneschi L, Matucci-Cerinic M. Endothelial-to-mesenchymal transition contributes to endothelial dysfunction and dermal fibrosis in systemic sclerosis. Ann Rheum Dis2017;76(5):924–934. <h3>Disclosure of Interest</h3> None declared