4th Department of Internal Medicine, Clinical Genomics and Pharmacogenomics Unit, 'Attikon' Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece; Molecular Biology Division, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
Laboratory of Biochemistry, University of Crete Medical School, Heraklion, Greece; Division of Gene Regulation and Genomics, Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas, Heraklion, Greece.
Metabolism. 2022 Feb;127:154954. doi: 10.1016/j.metabol.2021.154954. Epub 2021 Dec 4.
Atherosclerotic Coronary Artery Disease (ASCAD) is the leading cause of mortality worldwide. Novel therapeutic approaches aiming to improve the atheroprotective functions of High Density Lipoprotein (HDL) include the use of reconstituted HDL forms containing human apolipoprotein A-I (rHDL-apoA-I). Given the strong atheroprotective properties of apolipoprotein E3 (apoE3), rHDL-apoE3 may represent an attractive yet largely unexplored therapeutic agent.
To evaluate the atheroprotective potential of rHDL-apoE3 starting with the unbiased assessment of global transcriptome effects and focusing on endothelial cell (EC) migration as a critical process in re-endothelialization and atherosclerosis prevention. The cellular, molecular and functional effects of rHDL-apoE3 on EC migration-associated pathways were assessed, as well as the potential translatability of these findings in vivo.
Human Aortic ECs (HAEC) were treated with rHDL-apoE3 and total RNA was analyzed by whole genome microarrays. Expression and phosphorylation changes of key EC migration-associated molecules were validated by qRT-PCR and Western blot analysis in primary HAEC, Human Coronary Artery ECs (HCAEC) and the human EA.hy926 EC line. The capacity of rHDL-apoE3 to stimulate EC migration was assessed by wound healing and transwell migration assays. The contribution of MEK1/2, PI3K and the transcription factor ID1 in rHDL-apoE3-induced EC migration and activation of EC migration-related effectors was assessed using specific inhibitors (PD98059: MEK1/2, LY294002: PI3K) and siRNA-mediated gene silencing, respectively. The capacity of rHDL-apoE3 to improve vascular permeability and hypercholesterolemia in vivo was tested in a mouse model of hypercholesterolemia (apoE KO mice) using Evans Blue assays and lipid/lipoprotein analysis in the serum, respectively.
rHDL-apoE3 induced significant expression changes in 198 genes of HAEC mainly involved in re-endothelialization and atherosclerosis-associated functions. The most pronounced effect was observed for EC migration, with 42/198 genes being involved in the following EC migration-related pathways: 1) MEK/ERK, 2) PI3K/AKT/eNOS-MMP2/9, 3) RHO-GTPases, 4) integrin. rHDL-apoE3 induced changes in 24 representative transcripts of these pathways in HAEC, increasing the expression of their key proteins PIK3CG, EFNB2, ID1 and FLT1 in HCAEC and EA.hy926 cells. In addition, rHDL-apoE3 stimulated migration of HCAEC and EA.hy926 cells, and the migration was markedly attenuated in the presence of PD98059 or LY294002. rHDL-apoE3 also increased the phosphorylation of ERK1/2, AKT, eNOS and p38 MAPK in these cells, while PD98059 and LY294002 inhibited rHDL-apoE3-induced phosphorylation of ERK1/2, AKT and p38 MAPK, respectively. LY had no effect on rHDL-apoE3-mediated eNOS phosphorylation. ID1 siRNA markedly decreased EA.hy926 cell migration by inhibiting rHDL-apoE3-triggered ERK1/2 and AKT phosphorylation. Finally, administration of a single dose of rHDL-apoE3 in apoE KO mice markedly improved vascular permeability as demonstrated by the reduced concentration of Evans Blue dye in tissues such as the stomach, the tongue and the urinary bladder and ameliorated hypercholesterolemia.
rHDL-apoE3 significantly enhanced EC migration in vitro, predominantly via overexpression of ID1 and subsequent activation of MEK1/2 and PI3K, and their downstream targets ERK1/2, AKT and p38 MAPK, respectively, and improved vascular permeability in vivo. These novel insights into the rHDL-apoE3 functions suggest a potential clinical use to promote re-endothelialization and retard development of atherosclerosis.
动脉粥样硬化性冠状动脉疾病（ASCAD）是全球死亡的主要原因。旨在改善高密度脂蛋白（HDL）的抗动脉粥样硬化功能的新型治疗方法包括使用含有人载脂蛋白 A-I（rHDL-apoA-I）的重组 HDL 形式。鉴于载脂蛋白 E3（apoE3）的强大抗动脉粥样硬化特性，rHDL-apoE3 可能代表一种有吸引力但尚未充分探索的治疗药物。
通过对整体转录组效应的无偏评估，从 rHDL-apoE3 的抗动脉粥样硬化潜力开始，重点关注内皮细胞（EC）迁移作为再内皮化和动脉粥样硬化预防的关键过程。评估 rHDL-apoE3 对 EC 迁移相关途径的细胞、分子和功能影响，以及这些发现在体内的潜在转化。
用人主动脉内皮细胞（HAEC）处理 rHDL-apoE3，并通过全基因组微阵列分析总 RNA。使用实时定量 RT-PCR 和 Western blot 分析在原代 HAEC、人冠状动脉内皮细胞（HCAEC）和人 EA.hy926 内皮细胞系中验证关键 EC 迁移相关分子的表达和磷酸化变化。通过划痕愈合和 Transwell 迁移实验评估 rHDL-apoE3 刺激 EC 迁移的能力。使用特定抑制剂（PD98059：MEK1/2，LY294002：PI3K）和 siRNA 介导的基因沉默分别评估 MEK1/2、PI3K 和转录因子 ID1 在 rHDL-apoE3 诱导的 EC 迁移和激活 EC 迁移相关效应物中的作用。使用 Evans Blue 测定法和血清中的脂质/脂蛋白分析分别在高胆固醇血症（apoE KO 小鼠）模型中测试 rHDL-apoE3 改善血管通透性和高胆固醇血症的能力。
rHDL-apoE3 主要涉及再内皮化和动脉粥样硬化相关功能，诱导 HAEC 中 198 个基因的显著表达变化。最明显的影响是 EC 迁移，其中 42/198 个基因参与以下 EC 迁移相关途径：1）MEK/ERK，2）PI3K/AKT/eNOS-MMP2/9，3）RHO-GTPases，4）整合素。rHDL-apoE3 诱导 HAEC 中这些途径的 24 个代表性转录物的变化，增加了 HCAEC 和 EA.hy926 细胞中关键蛋白 PIK3CG、EFNB2、ID1 和 FLT1 的表达。此外，rHDL-apoE3 刺激 HCAEC 和 EA.hy926 细胞的迁移，并且在存在 PD98059 或 LY294002 的情况下，迁移明显减弱。rHDL-apoE3 还增加了这些细胞中 ERK1/2、AKT、eNOS 和 p38 MAPK 的磷酸化，而 PD98059 和 LY294002 分别抑制了 rHDL-apoE3 诱导的 ERK1/2、AKT 和 p38 MAPK 的磷酸化。LY 对 rHDL-apoE3 介导的 eNOS 磷酸化没有影响。ID1 siRNA 通过抑制 rHDL-apoE3 触发的 ERK1/2 和 AKT 磷酸化，显著降低了 EA.hy926 细胞的迁移。最后，apoE KO 小鼠单次给予 rHDL-apoE3 可显著改善血管通透性，表现为胃、舌和膀胱等组织中 Evans Blue 染料浓度降低，改善高胆固醇血症。
rHDL-apoE3 显著增强了体外 EC 迁移，主要通过过度表达 ID1 以及随后激活 MEK1/2 和 PI3K，及其下游靶标 ERK1/2、AKT 和 p38 MAPK，分别改善了体内血管通透性。这些对 rHDL-apoE3 功能的新见解表明，它可能具有促进再内皮化和延缓动脉粥样硬化发展的潜在临床用途。
