ABSTRACT
BACKGROUNDS/AIMS: Mesenchymal stromal cell-derived exosomes (MSC-EXs) could exert protective effects on recipient cells by transferring the contained microRNAs (miRs), and miR-132-3p is one of angiogenic miRs. However, whether the combination of MSC-EXs and miR-132-3p has better effects in ischemic cerebrovascular disease remains unknown. METHODS: Mouse MSCs transfected with scrambler control or miR-132-3p mimics were used to generate MSC-EXs and miR-132-3p-overexpressed MSC-EXs (MSC-EXsmiR-132-3p). The effects of EXs on hypoxia/reoxygenation (H/R)-injured ECs in ROS generation, apoptosis, and barrier function were analyzed. The levels of RASA1, Ras, phosphorylations of PI3K, Akt and endothelial nitric oxide synthesis (eNOS), and tight junction proteins (Claudin-5 and ZO-1) were measured. Ras and PI3K inhibitors were used for pathway analysis. In transient middle cerebral artery occlusion (tMCAO) mouse model, the effects of MSC-EXs on the cerebral vascular ROS production and apoptosis, cerebral vascular density (cMVD), Evans blue extravasation, brain water content, neurological deficit score (NDS), and infarct volume were determined. RESULTS: MSC-EXs could deliver their carried miR-132-3p into target ECs, which functionally downregulated the target protein RASA1, while upregulated the expression of Ras and the downstream PI3K phosphorylation. Compared to MSC-EXs, MSC-EXsmiR-132-3p were more effective in decreasing ROS production, apoptosis, and tight junction disruption in H/R-injured ECs. These effects were associated with increased levels of phosphorylated Akt and eNOS, which could be abolished by PI3K inhibitor (LY294002) or Ras inhibitor (NSC 23766). In the tMCAO mouse model, the infusion of MSC-EXsmiR-132-3p was more effective than MSC-EXs in reducing cerebral vascular ROS production, BBB dysfunction, and brain injury. CONCLUSION: Our results suggest that miR-132-3p promotes the beneficial effects of MSC-EXs on brain ischemic injury through protecting cerebral EC functions.
Subject(s)
Brain Injuries , Exosomes , Mesenchymal Stem Cells , MicroRNAs , Animals , Apoptosis , Brain , Exosomes/genetics , Mice , MicroRNAs/genetics , Phosphatidylinositol 3-Kinases/geneticsABSTRACT
Endothelial cells (ECs) released microvesicles (EMVs) could modulate the functions of target cells by transferring their microRNAs (miRs). We have reported that miR-125a-5p protected EC function. In this study, we determined whether EMVs provided beneficial effects on ECs by transferring miR-125a-5p. Human brain microvessel ECs were transfected with miR-125a-5p mimic or miR-125a-5p short hairpin RNA to obtain miR-125a-5p overexpressing ECs and miR-125a-5p knockdown ECs, and their derived EMVs. For the functional study, ECs or hypoxia/reoxygenation injured ECs were coincubated with various EMVs. The survival and angiogenic function of ECs were measured. Western blot and quantitative real time polymerase chain reaction (qRT-PCR) were used for measuring the levels of phosphoinositide 3-kinase (PI3K), phosphorylation-Akt (p-Akt)/Akt, p-endothelial nitric oxide synthase (p-eNOS), cleaved caspase-3, and miR-125a-5p. PI3K inhibitor was used for pathway analysis. EMVs promoted the proliferation, migration, and tube formation ability of ECs, and alleviated the apoptotic rate of ECs. These effects were associated by an increase in p-Akt/Akt and p-eNOS, and a decrease in cleaved caspase-3 could be abolished by LY294002. Overexpression or downregulation of miR-125a-5p in EMVs promoted or inhibited those effects of EMVs. EMVs could enhance the survival and angiogenic function of ECs via delivering miR-125a-5p to modulate the expression of PI3K/Akt/eNOS pathway and caspase-3.
Subject(s)
Endothelial Cells/metabolism , MicroRNAs/metabolism , Apoptosis/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Chromones/pharmacology , Coculture Techniques , Endothelial Cells/drug effects , Flow Cytometry , Humans , Morpholines/pharmacology , Nanoparticles/chemistry , Nitric Oxide Synthase Type III/metabolism , Particle Size , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/metabolism , Real-Time Polymerase Chain ReactionABSTRACT
Endothelial progenitor cells (EPCs) have shown the potential for treating ischemic stroke (IS), while microRNA-126 (miR-126) is reported to have beneficial effects on endothelial function and angiogenesis. In this study, we investigated the effects of miR-126 overexpression on EPCs and explore the efficacy of miR-126-primed EPCs (EPCmiR-126) in treating IS. The effects of miR-126 overexpression on EPC proliferation, migratory, tube formation capacity, reactive oxygen species (ROS) production, and nitric oxide (NO) generation were determined. In in vivo study, the effects of EPCmiR-126 on the cerebral blood flow (CBF), neurological deficit score (NDS), infarct volume, cerebral microvascular density (cMVD), and angiogenesis were determined. Moreover, the levels of circulating EPCs (cEPCs) and their contained miR-126 were measured. We found (1) miR-126 overexpression promoted the proliferation, migration, and tube formation abilities of EPCs; decreased ROS; and increased NO production of EPCs via activation of PI3K/Akt/eNOS pathway; (2) EPCmiR-126 was more effective than EPCs in attenuating infarct volume and NDS and enhancing cMVD, CBF, and angiogenesis; and (3) infusion of EPCmiR-126 increased the number and the level of miR-126 in cEPCs. Our data indicate that miR-126 overexpression enhanced the function of EPCs in vitro and in vivo.
