P681Blood coagulation factor Xa promotes the progression of atherosclerosis by enhancing inflammasome formation as a consequence of PAR2-mediated autophagy inhibition
Y. Maejima, Y. Ito, N. Tamura, M. Konishi, M.I. Isobe
European Heart Journal
plasma using ultracentrifugation. Flow cytometer and electron microscope were used for MP size characterization. Nine miRs involved in the regulation of vascular performance - miR-126, miR-222, miR-let7d, miR-21, miR-26, miR-92a, miR-133, miR-30 and miR-199a -were quantified in circulating MPs by qPCR. Among those, miR-92 was significantly increased in patients with CAD compared to non-CAD patients. MP-sorting experiments showed that endothelial cells (ECs) were the major cell source of MPs
... aining miR-92. A predominant packaging of miR-92 from ECs into EMPs was regulated by signal transducer and activator of transcription 3. Moreover, functional miR-92 can be exported via EMPs and affect adjacent ECs and VSMC functions. Knockdown of miR-92 in EMP abrogated EMP-mediated effects on HCAEC migration and proliferation and blocked vascular network formation in matrigel plug. Conclusion: Atherosclerotic conditions promote the sorting of endothelial miR-92 from ECs into EMPs. Intercellular transfer of functional miR-92 via EMPs on adjacent ECs regulate recipient cell biology with potential implications on health. Background: Increasing lines of evidence suggest that factor Xa (FXa), an endogenous blood coagulation factor, plays an important role in facilitating atherosclerosis, possibly through activating through protease-activated receptor-2 (PAR-2). However, the precise mechanism how FXa-mediated PAR-2 activation promotes atherogenesis remains to be elucidated. Purpose: The aim of this study is to explore how FXa promotes atherosclerosis through PAR-2-associated signaling pathway. Methods and results: Administration of direct FXa inhibitor rivaroxaban (Riv; 120 mg/kg/day) to the mice markedly elongated prothrombin time (Riv 120 mg/kg/day: 14.6±0.6*, Riv 60 mg/kg/day: 9.2±0.2*, Untreated: 7.1±0.1 sec, *P<0.05) and significantly suppressed the activity of plasma FXa compared with the mice treated with lower dose of Riv (60 mg/kg/day) or untreated mice (Riv 120 mg/kg/day: 1.9±0.1*, Riv 60mg/kg/day: 2.6±0.3*, Untreated: 3.9±0.3 RFU, *P<0.05). Treatment with Riv to ApoE knockout (KO) mice fed with high fat diet (HFD) significantly attenuated the area of atherosclerosis in the aorta compared with those in the untreated ApoE-KO mice fed with HFD as evaluated by Oil red-O staining (3.4% vs. 12.6%*, *P<0.05). The plaque size of ApoE-KO/PAR-2-KO bigeneic mice fed with HFD was similar to those of Riv-treated ApoE-KO mice fed with HFD (4.0% vs. 3.4%, N.S.). Ultrastructural examinations of atherosclerotic lesions by transmission electron microscopic (TEM) analyses revealed that the number of autophagosomes in the plaque-resident macrophages of Riv-treated ApoE-KO mice fed with HFD was significantly smaller than those of the untreated ApoE-KO mice fed with HFD. Immunostaining of NLRP3 revealed that Riv attenuated the inflammasome formation in the atherosclerotic lesion in ApoE-KO mice fed with HFD. In vitro experiments using murine macrophage cell lines or mouse peritoneal macrophages demonstrated that administration of 7-ketocholesterol (7KC) markedly enhanced autophagy activity as evidenced by the number of autophagosomes detected by TEM analyses and the increase in LC3-II protein. The addition of FXa (50 nM) significantly promoted mTOR (Ser2448) phosphorylation and blocked autophagy activity induced by 7KC, which was reversed in the presence of Riv (1 μM). Furthermore, immunoblot analyses using antibodies against NLRP3, caspase-1 and IL-1β demonstrated that FXa administration significantly accelerated inflammasome formation induced by 7KC, which was blocked in the presence of Riv. On the other hand, treatment with FXa failed to inhibit 7KC-induced autophagy and inflammasome activation in PAR-2-KO micederived macrophages. Conclusion: These results suggest that FXa aggravates atherogenesis through PAR-2-mediated pathway by inhibiting macrophage autophagy which, in turn, promoting inflammasome activation.