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

Abstract

Background & aims: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Lay summary: 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.

Keywords: Atherosclerosis; Extracellular vesicles; Inflammation; NAFLD; miRNA.