Department of Neurology, Guangdong Key Laboratory of Age-related Cardiac and Cerebral Diseases, Institute of Neurology, Affiliated Hospital of Guangdong Medical University, Zhanjiang, 524001, China.
Institute of Biochemistry and Molecular Biology, Guangdong Medical University, Zhanjiang, China.
Mol Neurobiol. 2023 Jun;60(6):3534-3552. doi: 10.1007/s12035-023-03280-4. Epub 2023 Mar 9.
Aging is one of the key mechanisms of vascular dysfunction and contributes to the initiation and progression of ischemic stroke (IS). Our previous study demonstrated that ACE2 priming enhanced the protective effects of exosomes derived from endothelial progenitor cells (EPC-EXs) on hypoxia-induced injury in aging endothelial cells (ECs). Here, we aimed to investigate whether ACE2-enriched EPC-EXs (ACE2-EPC-EXs) could attenuate brain ischemic injury by inhibiting cerebral EC damage through their carried miR-17-5p and the underlying molecular mechanisms. The enriched miRs in ACE2-EPC-EXs were screened using the miR sequencing method. EPC-EXs, ACE2-EPC-EXs, and ACE2-EPC-EXs with miR-17-5p deficiency (ACE2-EPC-EXs) were administered to transient middle cerebral artery occlusion (tMCAO)-operated aged mice or coincubated with hypoxia/reoxygenation (H/R)-treated aging ECs. The results showed that (1) the level of brain EPC-EXs and their carried ACE2 were significantly decreased in aged mice compared to in young mice, and (2) after tMCAO, aged mice displayed increases in brain cell senescence, infarct volume, and neurological deficit score (NDS) and a decrease in cerebral blood flow (CBF). (3) Compared with EPC-EXs, ACE2-EPC-EXs were enriched with miR-17-5p and more effective in increasing ACE2 and miR-17-5p expression in cerebral microvessels, accompanied by obvious increases in cerebral microvascular density (cMVD) and cerebral blood flow (CBF) and decreases in brain cell senescence, infarct volume, neurological deficit score (NDS), cerebral EC ROS production, and apoptosis in tMCAO-operated aged mice. Moreover, silencing of miR-17-5p partially abolished the beneficial effects of ACE2-EPC-EXs. (4) In H/R-treated aging ECs, ACE2-EPC-EXs were more effective than EPC-EXs in decreasing cell senescence, ROS production, and apoptosis and increasing cell viability and tube formation. In a mechanistic study, ACE2-EPC-EXs more effectively inhibited PTEN protein expression and increased the phosphorylation of PI3K and Akt, which were partially abolished by miR-17-5p knockdown. Altogether, our data suggest that ACE-EPC-EXs have better protective effects on ameliorating aged IS mouse brain neurovascular injury by inhibiting cell senescence, EC oxidative stress, apoptosis, and dysfunction by activating the miR-17-5p/PTEN/PI3K/Akt signaling pathway.
衰老是血管功能障碍的关键机制之一，也是缺血性中风（IS）发生和进展的原因。我们之前的研究表明，ACE2 预激活增强了内皮祖细胞（EPC）衍生的外泌体（EPC-EX）对缺氧诱导的衰老内皮细胞（EC）损伤的保护作用。在这里，我们旨在研究富含 ACE2 的 EPC-EX（ACE2-EPC-EX）是否可以通过抑制大脑 EC 损伤来减轻脑缺血损伤，其携带的 miR-17-5p 及其潜在的分子机制。使用 miR 测序方法筛选 ACE2-EPC-EX 中的富集 miR。将 EPC-EX、ACE2-EPC-EX 和 miR-17-5p 缺陷的 ACE2-EPC-EX（ACE2-EPC-EX）给予短暂性大脑中动脉闭塞（tMCAO）操作的老年小鼠或与缺氧/复氧（H/R）处理的衰老 EC 共孵育。结果表明：（1）与年轻小鼠相比，老年小鼠脑中 EPC-EXs 及其携带的 ACE2 水平明显降低；（2）tMCAO 后，老年小鼠表现出脑细胞衰老、梗死体积和神经功能缺损评分（NDS）增加，而脑血流（CBF）减少；（3）与 EPC-EXs 相比，ACE2-EPC-EXs 富含 miR-17-5p，并且在大脑微血管中更有效地增加 ACE2 和 miR-17-5p 的表达，伴随着大脑微血管密度（cMVD）和大脑血流（CBF）的明显增加，以及大脑细胞衰老、梗死体积、神经功能缺损评分（NDS）、大脑 EC ROS 产生和 tMCAO 操作后老年小鼠的凋亡减少；（4）在 H/R 处理的衰老 EC 中，ACE2-EPC-EXs 比 EPC-EXs 更有效地降低细胞衰老、ROS 产生和凋亡，增加细胞活力和管形成。在机制研究中，ACE2-EPC-EXs 更有效地抑制 PTEN 蛋白表达，增加 PI3K 和 Akt 的磷酸化，这部分被 miR-17-5p 敲低所阻断。总之，我们的数据表明，ACE-EPC-EXs 通过激活 miR-17-5p/PTEN/PI3K/Akt 信号通路抑制细胞衰老、EC 氧化应激、凋亡和功能障碍，对改善老年 IS 小鼠脑神经血管损伤具有更好的保护作用。
