ACE2-EPCs Protect Ecs Majorly Through Their Exosomal Effects on Mitochondria

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Angiotensin-converting enzyme 2 (ACE2) is an emerging cardiovascular protective target which mediates the metabolism of angiotensin (Ang) II into Ang (1-7). Our group has demonstrated that ACE2 overexpression enhances the therapeutic efficacy of endothelial progenitor cells (EPCs) for ischemic stroke. Here, we investigated whether ACE2-primed EPCs (ACE2-EPCs) can protect cerebral microvascular endothelial cells (ECs) against injury and dysfunction in an in vitro model, with focusing on their exosomal and cytokine paracrine effects on endothelial mitochondria. Human EPCs were transfected with lentivirus containing null or human ACE2 cDNA (denoted as Null-EPCs, ACE2-EPCs, respectively). Their conditioned culture media, w/wo ultra-centrifugation for depletion of exosomes (ACE2-EPC-CMEX-, Null-EPC-CMEX-, ACE2-EPC-CM and Null-EPC-CM), were used for co-culture experiments. ECs injury and dysfunction model was induced by Ang II (10-7 M) for 8 hrs before 16 hr co-culture. After that, ECs were collected for analyses of apoptosis, angiogenic ability, mitochondrion functions (ROS production, membrane potential, fragmentation), and gene expressions (ACE2, Nox2 and Nox4). The co-culture medium was collected for measuring the levels of ACE2, Ang II / Ang-(1-7) and growth factors (VEGF and IGF). Our results showed that: 1) ACE2-EPC-CM had higher levels of ACE2, Ang (1-7), VEGF and IGF than that of Null-EPC-CM. 2) Ang II-injured ECs displayed increase of apoptotic rate and reduction in tube formation and migration abilities, which were associated with ACE2 downregulation, Ang II / Ang (1-7) imbalance, Nox2/Nox4 upregulation, ROS overproduction, increase of mitochondrion fragmentation and decrease of membrane potential. 3) ACE2-EPC-CM had better protective effects than Null-EPC-CM on Ang II-injured ECs, which were associated with the improvements on ACE2 expression, Ang II / Ang (1-7) balance and mitochondrial functions. 4) ACE2-EPC-CMEX- and Null-EPC-CM EX- had remarkably reduced effects by over 70% as respectively compared to ACE2-EPCs-CM and Null-EPCs-CM. In conclusion, our data demonstrate that ACE2 overexpression can enhance the protective effects of EPCs on ECs injury, majorly through the exosomal effects on mitochondrial function.