P740Repeated social defeat exaggerates fibrin-rich clot formation in FeCl3-induced arterial thrombosis mice model by enhancing NETs formation

Background and objective Depression is an independent risk factor of cardiovascular disease (CVD). We have recently shown that repeated social defeat (RSD) precipitates depressive-like behaviorsin apoE−/− mice and exaggerates atherosclerosis development by enhancing neutrophil extracellular traps (NETs) formation (BBRC 2018; 500:490). Here, we investigated the impact of RSD on arterial thrombosis. Methods and results Eight-week-old male WT mice were exposed to RSDby housing with a larger CD-1 mouse in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. Control mice were housed in the same gage without physical contact. After social interaction testto confirm depressive-like behaviors, defeated mice (19 of 31) and control mice (12 of 14) were underwent arterial injury at 10 wks of age. A filter paper saturated with 10% FeCl3was applied on the adventitial surface of left carotid artery for 3 min and analyzed 3 hrs later. The volume of thrombi calculated by summing8–15 frozen cross-sectional images, each separated by 200 μm, was comparable between the 2 groups. However, fibrinogen/fibrin-positive areas in immunofluorescent images were significantly increased in defeated mice (27.8% vs. 48.8%, Control vs. Defeat, P<0.01).The numberof Ly-6G-positive cells in thrombi was markedly higher in defeated mice (144/mm2 vs. 878/mm2, Control vs. Defeat, P<0.05). Further, Ly-6G-positive cells were almost accumulated at the inner surface of injured artery, which were co-localized with neutrophil elastase, Cit-H3, and CD41-positive staining. Treatment with DNase Icompletely diminished the exaggerated fibrin-rich clot formation in defeated miceto a similar extent of control mice (25.7% vs. 22.3%, Control vs. Defeat, P= NS), while the volume of thrombi and number of Ly-6G-positive cells in thrombi were comparable between the 2 groups even afterDNase I treatment. Platelet aggregations induced by ADP or collagen were comparable between the 2 groups, suggesting that NETs formation primarily contributes to the exaggerated fibrin-rich clot formation in defeated mice. Conclusions Our findings demonstrate for the first time that repeated social defeat enhances fibrin-rich clot formation after arterial injury by enhancing NETs formation, suggesting that NETosis could be a new therapeutic target in depression-related CVD development.