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

• Published in: Annals of the rheumatic diseases Vol. 77; no. Suppl 2; p. 732
• Main Authors: Corallo, C., Benschop, J., Grimbergen, M., Crawford, J., Meuldijk, J., Janssen, R.A., Vandeghinste, N.E., Giordano, N., Tessari, M.A.
• Format: Journal Article
• Language: English
• Published: London BMJ Publishing Group LTD 01.06.2018
• Subjects: Cytokines; Drug discovery; Endothelial cells; Fibrosis; High-throughput screening; Immunocytochemistry; Mesenchyme; Microvasculature; Pathogenesis; Patients; Phenotypes; RNA-mediated interference; Scleroderma; Skin; Systemic sclerosis; Transforming growth factor-b
• ISSN: 0003-4967; 1468-2060
• DOI: 10.1136/annrheumdis-2018-eular.5406

BackgroundSystemic sclerosis (SSc) is an autoimmune connective tissue disease characterised by early vascular abnormalities and subsequent fibroblast activation and differentiation into myofibroblasts, leading to fibrosis.1,2 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.3 ObjectivesWe developed a high-content screening assay with the aim to identify novel proteins which, upon inhibition, will reduce EndoMT in SSc.MethodsHuman 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.ResultsWe 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.ConclusionsThis 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.References[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.Disclosure of InterestNone declared