The hallmark of atherogenesis is characterized as endothelial dysfunction and subsequent macrophage activation. Although our previous study has demonstrated that endothelin-1 (ET-1) plays an important role in atherogenesis, the underlying mechanism remains deeply investigation. Enhanced atherosclerotic plaques were observed in endothelium-specific ET-1 overexpression ApoE^−/- mice (eET-1/ApoE^−/-) concomitant with increased secretion of pro-inflammatory adhesion molecules and cytokines. The conditional media used for culturing human umbilical vein endothelial cells (HUVECs) with AdET-1 infection and subjected to OX-LDL stimulation, was collected and utilized for bone marrow-derived macrophages (BMDMs) culturing. RT-PCR analysis showed increased genes expression related to classical M1 macrophages but decreased alternative activated M2 macrophages genes expression in macrophage culturing with the conditional media. Furthermore, consistent regulations of macrophage polarization were observed using isolated exosomes from the conditional media. More importantly, we noticed that miR-33 was enriched in the exosomes derived by HUVECs with AdET-1 infection, while bioinformatics analysis further indicated that miR-33 directly targeted NR4A and miR-33/NR4A axis was required for the effect of endothelial-specific ET-1 overexpression on pro-inflammatory macrophage activation. By contrast, such effects could be reversed by ET-1 knockdown. Taken together, our study indicated that the exosomes derived by HUVECs with AdET-1 infection can transfer miR-33 to macrophages and subsequently promote pro-inflammatory macrophage activation by directly targeting to NR4A. These evidences clearly revealed that miR-33/NR4A axis was the important mechanism underlying the effect of ET-1 on macrophage activation and indicated that ET-1 may act as a promising target for atherosclerosis management.

动脉粥样硬化的特征是内皮功能障碍和随后的巨噬细胞活化。尽管我们先前的研究表明内皮素-1（ET-1）在动脉粥样硬化中起重要作用，但其潜在机制仍需深入研究。在内皮特异性ET-1过表达ApoE ^-/-小鼠（eET-1 / ApoE ^-/-）中观察到动脉粥样硬化斑块增强）伴随促炎性粘附分子和细胞因子分泌的增加。收集用于培养受AdET-1感染并经过OX-LDL刺激的人脐静脉内皮细胞（HUVEC）的条件培养基，并将其用于骨髓源巨噬细胞（BMDM）培养。RT-PCR分析显示，在有条件培养基中培养的巨噬细胞中，与经典M1巨噬细胞相关的基因表达增加，而替代的活化M2巨噬细胞基因表达则下降。此外，使用从条件培养基分离的外泌体观察到巨噬细胞极化的一致规律。更重要的是，我们注意到miR-33富含由AdVE-1感染的HUVEC衍生的外来体，生物信息学分析进一步表明，直接针对NR4A和miR-33 / NR4A轴的miR-33是内皮特异性ET-1过表达对促炎性巨噬细胞激活的影响所必需的。相比之下，这种效应可以通过ET-1敲低来逆转。综上所述，我们的研究表明，HUVEC衍生的AdET-1感染的外泌体可以将miR-33转移至巨噬细胞，并随后通过直接靶向NR4A来促进促炎性巨噬细胞活化。这些证据清楚地表明，miR-33 / NR4A轴是ET-1影响巨噬细胞活化的重要机制，并表明ET-1可能成为动脉粥样硬化治疗的有希望的靶标。相比之下，这种效应可以通过ET-1敲低来逆转。综上所述，我们的研究表明，HUVEC衍生的AdET-1感染的外泌体可以将miR-33转移至巨噬细胞，并随后通过直接靶向NR4A来促进促炎性巨噬细胞活化。这些证据清楚地表明，miR-33 / NR4A轴是ET-1影响巨噬细胞活化的重要机制，并表明ET-1可能成为动脉粥样硬化治疗的有希望的靶标。相比之下，这种效应可以通过ET-1敲低来逆转。综上所述，我们的研究表明，HUVEC衍生的AdET-1感染的外泌体可以将miR-33转移至巨噬细胞，并随后通过直接靶向NR4A来促进促炎性巨噬细胞活化。这些证据清楚地表明，miR-33 / NR4A轴是ET-1影响巨噬细胞活化的重要机制，并表明ET-1可能成为动脉粥样硬化治疗的有希望的靶标。