Coxsackievirus B3 decreased angiogenic activity of endothelial colony forming cells through TLR3 activation

MENA, HA; SCHATTNER, M; ONETO, P; GÓMEZ, R; ZUBIRY, P; ETULAIN, J; NEGROTTO, S

Melbourne

Congreso; XXVII Congress of the International Society on Thrombosis and Haemostasis (ISTH); 2019

International Society on Thrombosis and Haemostasis (ISTH)

Background: Coxsackievirus B3 (CVB3), a ssRNA Enterovirus, is the main cause of humanviral myocarditis and dilated cardiomyopathy. Local or homed progenitor cells are involved inmyocardial repair, especially endothelial progenitor cells, which have a lead role in tissuerevascularization. We have previously demonstrated that CVB3 infects and replicates in lateoutgrowth endothelial colony forming cells (ECFC) and significantly reduced their angiogenicresponses.Aims: We h ere aimed to study the receptors and molecular pathways involved in CVB3-mediated antiangiogenic effects.Methods: Human cord blood-de rived CD34 cells were seeded in EGM2 and ECFC colonieswere obtained after 14-21 days. ECFC were incubated with CVB3 at the indicated multiplicity ofinfection (MOI) for 1 h. N=3-5. *p< 0.05 vs Mock (M) or untreated control (C), #p< 0.05 vs MOI0.1, ANOVA.Results: Flow cytometric studies indicated that Coxsackie and Adenovirus Receptor (CAR) isexpressed in ECFC and upregulated by infection (M: 8±1; MOI 0.1: 13±2; MOI 1: 14±2; MOI 10:21±2* % of CAR+ cells). The ssRNA sensors TLR7 and TLR8 were not detected (RT-qPCR). Infact, TLR7 and TLR8 agonists CL264, TL8-506 and Imiquimod failed to modify ECFC lifespan.In contrast, TLR3 expression, which can detect dsRNA generated during viral replication, isexpressed by ECFC and upregulated by CVB3 infection (RT-qPCR, Figure 1). Similar resultswere obtained with TLR3 downstream molecules IRF7 and INFβ, but not IRF3 (Figure 1).Moreover, CVB3-mediated impairment of proliferation (cell count) and tubulogenesis (matrigel)were completely prevented by the TLR3 inhibitor 4a (27 μM) and the CVB3 antiangiogeniceffects were mimicked by two TLR3 agonists like poly(I:C) and poly(A:U) (100 μg/ml) (Figure 2).Conclusions: In conclusion, CVB3 infection downregulates ECFC angiogenic responsesthrough TLR3 activation, pointing out endothelial TLR3 blockade as a possible therapeuticapproach to restore ECFC angiogenic properties and the consequent increase in myocardialrepair.