Endothelial extracellular vesicles modulate the macrophage phenotype: Potential implications in atherosclerosis.

Endothelial cells (ECs) and macrophages engage in tight and specific interactions that play critical roles in cardiovascular homeostasis and the pathogenesis of atherosclerosis. Extracellular vesicles (EVs) are circular membrane fragments released from the endosomal compartment as exosomes or shed from the surfaces of the membranes of most cell types. Increasing evidence indicates that EVs play a pivotal role in cell-to-cell communication. However, the contribution of EVs, as determine by oxidized low-density lipoprotein (ox-LDL)-exposed and/or Kruppel-like factor 2 (KLF2)-transduced ECs in the interaction between vascular ECs and monocytes/macrophages, which is a key event in atherosclerotic plaque development, has remained elusive. This study demonstrates the characteristic impact of EVs from ox-LDL-treated and/or KLF2-transduced ECs on the monocyte/macrophage phenotype in vitro and in vivo.Q-PCR showed that both the atherosclerosis inducer ox-LDL and atheroprotective factor KLF2 regulated inflammation-associated microRNA-155 (miR-155) expression in human umbilical vein endothelial cells (HUVECs). Moreover, coculture, immunofluorescence and flow cytometry revealed that miR-155 was enriched in ox-LDL-induced ECs-EVs and subsequently transferred to human monocytic THP1 cells, in which these vesicles enhance monocyte activation by shifting the monocytes/macrophages balance from anti-inflammatory M2 macrophages towards proinflammatory M1 macrophages; EVs from KLF2-expressing ECs suppressed monocyte activation by enhancing immunomodulatory responses and diminishing proinflammatory responses, which indicate the potent anti-inflammatory activities of these cells. Furthermore, oil red staining showed that atherosclerotic lesions were reduced in mice that received EVs from KLF2-transduced ECs with decreased proinflammatory M1 macrophages and increased anti-inflammatory M2 macrophages, and this effect is at least partly due to the decreased expression of inflammation-associated miR-155, confirming our in vitro findings. In summary, this study provides novel insights into the pathophysiological effects of altered EV secretion and/or microRNA content and their influence on modulating monocyte activation depending on the environment surrounding EVs-releasing ECs.

He S ，Wu C ，Xiao J ，Li D ，Sun Z ，Li M ... -  《-》

Extracellular vesicles derived from Krüppel-Like Factor 2-overexpressing endothelial cells attenuate myocardial ischemia-reperfusion injury by preventing Ly6C(high) monocyte recruitment.

Background: The ischemia/reperfusion (I/R) process in patients with ST-segment elevation myocardial infarction (STEMI) triggers an immune response, resulting in myocyte death. Krüppel-Like Factor 2 (KLF2), which is highly expressed in endothelial cells (ECs) under laminar flow, exerts anti-inflammatory effects. In this study, we explored the role of small extracellular vesicles (EVs) from KLF2-overexpressing ECs (KLF2-EVs) in the immunomodulation and its implications in myocardial I/R injury. Methods and Results: The small EVs were isolated from KLF2-overexpressing ECs' supernatant using gradient centrifugation. Mice were subjected to 45 min of ischemia followed by reperfusion, and KLF2-EVs were administrated through intravenous injection. KLF2-EVs ameliorated I/R injury and alleviated inflammation level in the serum and heart. We employed the macrophage depletion model and splenectomy and showed that Ly6Chigh monocyte recruitment from bone marrow was the main target of KLF2-EVs. miRNA-sequencing of KLF2-EVs and bioinformatics analysis implicated miRNA-24-3p (miR-24-3p) as a potent candidate mediator of monocyte recruitment and CCR2 as a downstream target. miR-24-3p mimic inhibited the migration of Ly6Chigh monocytes, and miR-24-3p antagomir reversed the effect of KLF2-EVs in myocardial I/R. Conclusion: Our data demonstrated that KLF2-EVs attenuated myocardial I/R injury in mice via shuttling miR-24-3p that restrained the Ly6Chigh monocyte recruitment. Thus, KLF2-EVs could be a potential therapeutic agent for myocardial I/R injury.

Qiao S ，Zhang W ，Yin Y ，Wei Z ，Chen F ，Zhao J ，Sun X ，Mu D ，Xie J ，Xu B ... -  《Theranostics》

Kruppel-Like Factor 2-Mediated Suppression of MicroRNA-155 Reduces the Proinflammatory Activation of Macrophages.

He S ，Yang L ，Li D ，Li M ... -  《PLoS One》

Hepatocyte-derived extracellular vesicles promote endothelial inflammation and atherogenesis via microRNA-1.

Clinical evidence has indicated a close link between non-alcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD). However, the underlying mechanism remains to be elucidated. This study aimed to explore a potential role of hepatocyte-derived extracellular vesicles (EVs) in endothelial inflammation and atherogenesis in the context of NAFLD. EVs were isolated, quantified and characterized from steatotic hepatocytes. An endothelial cell-specific PCR array was used to screen the functional properties of EVs. Profiling of global microRNA expression was conducted in EVs. The expression level and biological function of microRNA-1 (miR-1) was determined by quantitative PCR, immunoblot and reporter gene assays, respectively. The in vivo effect of miR-1 on atherogenesis was investigated in apolipoprotein E (ApoE)-deficient mice administered with a miR-1-specific inhibitor, antagomiR-1. Steatotic hepatocytes released more EVs, which had significantly altered miRNA expression profiles compared to the EVs released by control hepatocytes. Endothelial cells co-cultured with steatotic hepatocytes, or treated with their EVs or miR-1, expressed significantly more proinflammatory molecules, as well as exhibiting increased NF-κB activity and reduced Kruppel-like factor 4 (KLF4) expression. EV-induced endothelial inflammation was prevented by either downregulation or inhibition of miR-1. While miR-1 treatment suppressed KLF4 expression and reporter gene activity, overexpression of KLF4 dramatically abolished the miR-1-induced endothelial inflammation. Moreover, not only did the miR-1 inhibitor reduce endothelial inflammation in vitro, but it also attenuated atherogenesis in ApoE-deficient mice. Steatotic hepatocyte-derived EVs promote endothelial inflammation and facilitate atherogenesis by miR-1 delivery, KLF4 suppression and NF-κB activation. The findings illustrate an important role of hepatocyte-derived EVs in distant communications between the liver and vasculature, suggesting a new mechanism underlying the link between NAFLD and CVD. Non-alcoholic fatty liver disease (NAFLD), a condition highly prevalent in obese and/or diabetic patients, is emerging as an independent risk factor of cardiovascular disease. Herein, we demonstrated that extracellular vesicles, released by hepatocytes under NAFLD conditions, cause vascular endothelial inflammation and promote atherosclerosis. Within these toxic vesicles, we identified a small molecular cargo that acted as a potent inducer of endothelial inflammation. By inhibiting this cargo's function, a specific gene-based inhibitor profoundly attenuated atherogenesis in mice, uncovering a novel mechanism which may be used to prevent or treat cardiovascular disease in patients with NAFLD.

Jiang F ，Chen Q ，Wang W ，Ling Y ，Yan Y ，Xia P ... -  《-》

Extracellular MicroRNA-92a Mediates Endothelial Cell-Macrophage Communication.

Chang YJ ，Li YS ，Wu CC ，Wang KC ，Huang TC ，Chen Z ，Chien S ... -  《-》