Abstract Body: Introduction: COVID19-associated immunopathology is associated with increased production of interferon (IFN)-alpha (IFNα) and lambda3 (IFNL3). Effects of IFNs are mediated by interferon-stimulated genes (ISGs) and influence expression of angiotensin-converting enzyme 2 (ACE2), the receptor for S-protein (S1P) of SARS-CoV-2. We hypothesized that S1P-induced immune/inflammatory responses in endothelial cells (EC) are mediated via IFNs and may be important in vascular dysfunction associated with hypertension.
Methods: Human microvascular ECs (MEC) were stimulated with S1P (1µg/mL), IFNα (100ng/mL) or IFNL3 (100IU/mL). Because ACE2, metalloproteinase domain-17 (ADAM17) and type-II transmembrane serine protease (TMPRSS2) are important for SARS-CoV-2 infection, we used inhibitors of ADAM17 (marimastat), ACE2 (MLN4760), and TMPRSS2 (camostat). Gene and protein expression was investigated by real-time PCR and immunoblotting. Vascular function was assessed in mesenteric arteries from wild-type (WT) normotensive and hypertensive mice and in ISG15KO mice.
Results: MEC stimulated with S1P increased expression of IFNα (3-fold), IFNL3 (4-fold) and ISGs (2-fold) (p<0.05). IFNα and IFNλ3 increased protein expression of TMPRSS2 and ADAM17, but not ACE2. MEC exhibited higher responses to IFNα (ISG15: 16-fold) than to IFNL3 (ISG15: 1.7-fold) (p<0.05). ISG15 was also increased by S1P, effects that were enhanced by MLN4760 (2-fold) and reduced by marimastat (50%). S1P increased IL-6 mRNA (1.3-fold), TNFα (6.2-fold) and IL-1β (3.3-fold). These responses were increased by by IFNs. IL-6 was increased by IFNα (1,230pg/mL) and IFNL3 (1,124pg/mL) vs control (591pg/mL). This was associated with increased phosphorylation of Stat1 (134%), Stat2 (102%), ERK1/2 (42%). Nitric oxide production and eNOS phosphorylation (Ser1177) were reduced by IFNα and (40%) and IFNL3 (40%). Reduced endothelium-relaxation maximal response (%Emax) was observed in vessels from WT-mice stimulated with IFNα (67%) and IFNL3 (71%) vs control (82%) (p<0.05) but not in vessels from ISG15KO mice. Increased contraction was observed only in vessels from hypertensive mice treated with IFNα (9.1±0.5mN vs control: 7.3±0.3mN, p<0.05).
Conclusions: In ECs, S1P, IFNα and IFNL3 increased ISG15 and IL-6, processes that involve ADAM17 and are independent on ACE2. Inflammation induced by S1P was amplified by IFNs. IFNs induce vascular dysfunction through ISG15-dependent mechanisms, with augmented effects in hypertension.