THU0022 ETANERCEPT INCREASES AUTOPHAGY AND REDUCES APOPTOSIS INDUCED BY TNF-αLPHA EXPOSURE IN ENDOTHELIAL CELLS IN VITRO
Authors: Alessandra Ida Celia, Cristiana Barbati, Fulvia Ceccarelli, Tania Colasanti, Annacarla Finucci, Carlo Perricone, Mariangela Speziali, Francesca Romana Spinelli, Marta Vomero, Fabrizio Conti, Guido Valesini, Cristiano Alessandri
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> Rheumatoid arthritis (RA) is associated with high incidence of cardiovascular events, mainly due to accelerated atherosclerosis and endothelial dysfunction (1). Apoptosis in endothelial cells has been associated with vascular wall damage, contributing to atherosclerotic plaque formation (2). At the contrary, autophagy may play a protective role in preventing development of atherosclerosis. However, the exact mechanism which controls the autophagy of endothelial cells is still unknown. Tumor necrosis factor alpha (TNF-α) is one of the leading cytokines involved in the pathogenesis of RA. Several evidences suggest a pro-atherogenic effect of TNF-α in patients with RA. Etanercept (ETA) and other TNF-α inhibitors are effective treatments for joint inflammation in RA, but their effect on endothelial dysfunction is still not well understood. <h3>Objectives:</h3> The aim of this study was to investigate in <i>vitro</i>, the effects of ETA treatment on endothelial cells exposed to TNF-α, with particular attention on apoptosis levels and autophagy pathway. <h3>Methods:</h3> <i>In vitro</i> effects of ETA and TNF-α on endothelium were evaluated using human umbilical vein cell line EA.hy926. Cells were treated with ETA (15μg), TNF-α (10 ng/ml), alone or in combination. An untreated control cell culture was also performed. After 24 hours, apoptosis levels and autophagy have been evaluated. Apoptosis was analyzed by flow cytometry using a FITC-conjugated annexin V and propidium iodide apoptosis detection kit (3); autophagy was analyzed by western blot for the expression level of the autophagic markers LC3-II and p-62 (4). <h3>Results:</h3> TNF-α treatment significantly increased apoptosis in endothelial cells compared both to untreated and ETA-treated cells (p= 0.0067 and p= 0.00187 respectively) [Figure 1]. The co-treatment with TNF-α and ETA significantly reverted the increase of apoptosis levels (p= 0.001 versus TNFα alone). Both ETA and TNFα treatments significantly increased the expression level of LC3II in treated cells when compared to untreated cells (p=.18 and p=.3 respectively). The co-treatment induced an additive effect on autophagy levels (p&lt; 0.0001 versus untreated cells). At last, the expression level of p-62 showed an inversely proportional trend to LC3II levels [ETA (p=0.0001), TNF-α p=.1), ETA+TNF-α p=5) versus untreated cells, respectively] [Figure 2]. <h3>Conclusion:</h3> Our results show that treatment with Etanercept have a protective effect on endothelial cells <i>in vitro</i>, reversing the apoptosis induced by TNF-α. Moreover, this is the first study that supports a possible effect of Etanercept in increasing autophagy level in endothelial cells. Reducing TNFα-induced apoptosis, Etanercept treatment may lead to an improvement of endothelial function and to a reduction of accelerated atherosclerosis. <h3>References:</h3> [1] Hürlimann D, Forster A, Noll G, Enseleit F, Chenevard R, Distler O, et al. Anti-tumor necrosis factor-a treatment improves endothelial function in patients with rheumatoid arthritis. Circulation. 2002;106:2184–2187 [2] Fukuo K, Nakahashi T, Nomura S, Hata S, Suhara T, Shimizu M, et al. Possible participation of Fas-mediated apoptosis in the mechanism of atherosclerosis. Gerontology. 1997;1:35–42. [3] Alessandri C, Barbati C, Vacirca D, Piscopo P, Confaloni A, Sanchez M, et al. T lymphocytes from patients with systemic lupus erythematosus are resistant to induction of autophagy. FASEB J. 2012;26:4722–4732. [4] Barbati C, Alessandri C, Vomero M, Vona R, Colasanti T, Vacirca D, et al. Autoantibodies specific to D4GDI modulate Rho GTPase mediated cytoskeleton remodeling and induce autophagy in T lymphocytes. J Autoimmun. 2015;58:78–89. <h3>Disclosure of Interests:</h3> None declared