Ji Yang, Chen Huanzhen, Pang Lihua, Chen Changnong, Wang Sha, Chen Jing, Fang Lei, Liu Benrong, Cheng Yongruo, Liu Shiming, Zhong Yun
Guangzhou Institute of Cardiovascular Disease, Guangdong Key Laboratory of Vascular Diseases, State Key Laboratory of Respiratory Disease, The Second Afliated Hospital, Guangzhou Medical University, Guangzhou, 510260, Guangdong, China.
Department of Emergency, The Second Affliated Hospital, Guangzhou Medical University, Guangzhou, 510260, Guangdong, China.
Cardiovasc Diabetol. 2025 Apr 9;24(1):158. doi: 10.1186/s12933-025-02715-7.
Endothelial dysfunction is a pivotal contributor to cardiovascular complications in individuals with diabetes. However, the precise role of macrophages and their exosomes in the diabetic milieu remains elusive.
Exosomes (Exos) were isolated from the supernatants of macrophages treated with advanced glycation end products (AGE) or bovine serum albumin (BSA) using ultracentrifugation. Following coculture with AGE-Exos or BSA-Exos, human umbilical vein endothelial cells (HUVECs) were subjected to CCK-8, EdU, cell migration, monocyte adhesion, and tube formation assays. ELISA and Western blotting were employed to assess inflammatory cytokine release and protein expression levels in HUVECs. The miRNA expression profiles of AGE-Exos and BSA-Exos were analysed using miRNA arrays. Potential targets of miR-22-5p were predicted via miRNA databases and validated through RT‒qPCR, dual-luciferase reporter assays, and rescue experiments. Furthermore, a Rab27a knockout mouse model of type 2 diabetes mellitus (T2DM) was established by intraperitoneal injection of Streptozotocin. Aortic tissues were analysed via immunofluorescence for CD63 and CD31 expression, immunohistochemistry for VCAM-1 and ICAM-1 expression, and Western blotting for FOXP1 expression.
AGE stimulation increased the secretion of exosomes from macrophages. Compared with BSA-Exos, AGE-Exos significantly impaired endothelial cell proliferation, migration, and tube formation capabilities while increasing monocyte adhesion and proinflammatory cytokine release without affecting cell viability. miR-22-5p was enriched in AGE-Exos, which were subsequently transferred to HUVECs, specifically targeting FOXP1, resulting in endothelial dysfunction. Overexpression of miR-22-5p in HUVECs using lentiviral vectors recapitulated the inflammatory effects observed with AGE-Exos, whereas anti-miR-22-5p conferred protective effects. Rab27a knockout significantly reduced exosome accumulation in T2DM model mouse aortic tissues, alleviating endothelial discontinuity, downregulating VCAM-1 and ICAM-1 expression, and upregulating FOXP1 expression.
AGE-induced release of macrophage-derived exosomes may partially depend on Rab27a transport, which delivers miR-22-5p to ECs. This miR-22-5p targets FOXP1 in ECs, leading to inflammation and resulting in endothelial dysfunction that accelerates the development of diabetic vascular lesions.
内皮功能障碍是糖尿病患者心血管并发症的关键促成因素。然而，巨噬细胞及其外泌体在糖尿病环境中的精确作用仍不清楚。
使用超速离心法从用晚期糖基化终产物（AGE）或牛血清白蛋白（BSA）处理的巨噬细胞的上清液中分离外泌体（Exos）。在与AGE-Exos或BSA-Exos共培养后，对人脐静脉内皮细胞（HUVECs）进行CCK-8、EdU、细胞迁移、单核细胞黏附及管形成实验。采用酶联免疫吸附测定（ELISA）和蛋白质免疫印迹法评估HUVECs中炎性细胞因子的释放及蛋白质表达水平。使用miRNA芯片分析AGE-Exos和BSA-Exos的miRNA表达谱。通过miRNA数据库预测miR-22-5p的潜在靶标，并通过逆转录定量聚合酶链反应（RT-qPCR）、双荧光素酶报告基因检测及拯救实验进行验证。此外，通过腹腔注射链脲佐菌素建立2型糖尿病（T2DM）的Rab27a基因敲除小鼠模型。通过免疫荧光分析主动脉组织中CD63和CD31的表达，免疫组织化学分析血管细胞黏附分子-1（VCAM-1）和细胞间黏附分子-1（ICAM-1）的表达，蛋白质免疫印迹法分析叉头框蛋白P1（FOXP1）的表达。
AGE刺激增加了巨噬细胞外泌体的分泌。与BSA-Exos相比，AGE-Exos显著损害内皮细胞的增殖、迁移及管形成能力，同时增加单核细胞黏附和促炎细胞因子释放，而不影响细胞活力。miR-22-5p在AGE-Exos中富集，随后转移至HUVECs，特异性靶向FOXP1，导致内皮功能障碍。使用慢病毒载体在HUVECs中过表达miR-22-5p重现了AGE-Exos所观察到的炎性效应，而抗miR-22-5p则具有保护作用。Rab27a基因敲除显著减少了T2DM模型小鼠主动脉组织中外泌体的积累，减轻了内皮细胞的连续性破坏，下调了VCAM-1和ICAM-1的表达，并上调了FOXP1的表达。
AGE诱导的巨噬细胞来源外泌体的释放可能部分依赖于Rab27a转运，其将miR-22-5p传递至内皮细胞。该miR-22-5p靶向内皮细胞中的FOXP1，导致炎症反应，进而引起内皮功能障碍，加速糖尿病血管病变的发展。
