Badimon Lina, Luquero Aureli, Crespo Javier, Peña Esther, Borrell-Pages Maria
CIBER-CV, Instituto de Salud Carlos III, Spain.
Cardiovascular Research Chair, UAB, Barcelona, Spain.
Cardiovasc Res. 2021 Jul 27;117(9):2054-2068. doi: 10.1093/cvr/cvaa254.
Atherosclerosis, the leading cause of cardiovascular diseases, is driven by high blood cholesterol levels and chronic inflammation. Low-density lipoprotein receptors (LDLR) play a critical role in regulating blood cholesterol levels by binding to and clearing LDLs from the circulation. The disruption of the interaction between proprotein convertase subtilisin/kexin 9 (PCSK9) and LDLR reduces blood cholesterol levels. It is not well known whether other members of the LDLR superfamily may be targets of PCSK9. The aim of this work was to determine if LDLR-related protein 5 (LRP5) is a PCSK9 target and to study the role of PCSK9 and LRP5 in foam cell formation and lipid accumulation.
Primary cultures of human inflammatory cells (monocytes and macrophages) were silenced for LRP5 or PCSK9 and challenged with LDLs. We first show that LRP5 is needed for macrophage lipid uptake since LRP5-silenced macrophages show less intracellular CE accumulation. In macrophages, internalization of LRP5-bound LDL is already highly evident after 5 h of LDL incubation and lasts up to 24 h; however, in the absence of both LRP5 and PCSK9, there is a strong reduction of CE accumulation indicating a role for both proteins in lipid uptake. Immunoprecipitation experiments show that LRP5 forms a complex with PCSK9 in lipid-loaded macrophages. Finally, PCSK9 participates in TLR4/NFkB signalling; a decreased TLR4 protein expression levels and a decreased nuclear translocation of NFκB were detected in PCSK9 silenced cells after lipid loading, indicating a downregulation of the TLR4/NFκB pathway.
Our results show that both LRP5 and PCSK9 participate in lipid uptake in macrophages. In the absence of LRP5, there is a reduced release of PCSK9 indicating that LRP5 also participates in the mechanism of release of soluble PCSK9. Furthermore, PCSK9 up-regulates TLR4/NFκB favouring inflammation.
动脉粥样硬化是心血管疾病的主要病因，由高血胆固醇水平和慢性炎症驱动。低密度脂蛋白受体（LDLR）通过与循环中的低密度脂蛋白（LDL）结合并清除LDL，在调节血胆固醇水平中起关键作用。前蛋白转化酶枯草杆菌蛋白酶/kexin 9型（PCSK9）与LDLR之间相互作用的破坏可降低血胆固醇水平。LDLR超家族的其他成员是否可能是PCSK9 的作用靶点尚不清楚。这项工作的目的是确定低密度脂蛋白受体相关蛋白5（LRP5）是否为PCSK9 的作用靶点，并研究PCSK9 和LRP5 在泡沫细胞形成和脂质积累中的作用。
对人炎症细胞（单核细胞和巨噬细胞）的原代培养物进行LRP5 或PCSK9 基因沉默，并用LDL 进行刺激。我们首先表明，巨噬细胞摄取脂质需要LRP5，因为LRP5 基因沉默的巨噬细胞显示细胞内胆固醇酯（CE）积累较少。在巨噬细胞中，与LRP5 结合的LDL 的内化在LDL 孵育5 小时后已经非常明显，并持续长达24 小时；然而，在同时缺乏LRP5 和PCSK9 的情况下，CE 积累显著减少，表明这两种蛋白在脂质摄取中均起作用。免疫沉淀实验表明，在脂质负载的巨噬细胞中，LRP5 与PCSK9 形成复合物。最后，PCSK9 参与Toll 样受体4（TLR4）/核因子κB（NFκB）信号传导；脂质负载后，在PCSK9 基因沉默的细胞中检测到TLR-4 蛋白表达水平降低和NFκB 的核转位减少，表明TLR4/NFκB 通路下调。
我们的结果表明，LRP5 和PCSK9 均参与巨噬细胞的脂质摄取。在缺乏LRP5 的情况下，PCSK9 的释放减少，表明LRP5 也参与可溶性PCSK9 的释放机制。此外，PCSK9 上调TLR4/NFκB，促进炎症反应。
