Upregulation of Extracellular Vesicles-Encapsulated miR-132 Released From Mesenchymal Stem Cells Attenuates Ischemic Neuronal Injury by Inhibiting Smad2/c-jun Pathway via Acvr2b Suppression

Abstract

Ischemic cerebrovascular disease is a significant and common public health issue worldwide. The emerging roles of mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) in ischemic neuronal injury continue to be investigated. The current study aimed to investigate the role of EV-derived miR-132 from MSCs in ischemic neuronal injury. EVs were initially isolated from bone MSCs (BMSCs) and subsequently evaluated. A middle cerebral artery occlusion (MCAO) mouse model was constructed with the neurological function evaluated through a series of neurological scores, a pole test, and a foot fault test. Histopathological changes, neuron viability, and apoptosis, as well as cerebral infarction, were detected by hematoxylin and eosin (HE) staining and 2,3,5-triphenyltetrazolium hydrochloride (TTC) staining. The targeting relationship between microRNA (miR)-132 and Activin receptor type IIB (Acvr2b) was further confirmed based on dual-luciferase reporter gene assay results. Loss- and gain-of-function assays were conducted to elucidate the role of miR-132, EV-derived miR-132, Acvr2b, and Smad2 in oxygen-glucose deprivation (OGD)-treated neurons, and in mice models. Neuronal cell viability and apoptosis were evaluated via Cell Counting kit-8 (CCK-8) and flow cytometry. Our results indicated that Acvr2b was highly expressed, while miR-132 was poorly expressed in the MCAO mice and OGD-treated neurons. Acvr2b silencing or upregulation of miR-132 led to an elevation in neuronal activity, decreased neuronal apoptosis, reduced expression of Bax, and cleaved-caspase 3, as well as increased Bcl-2 expression. Acvr2b expression was targeted and inhibited by miR-132. EV-derived Acvr2b promoted activation of phosphorylated-Smad2 (p-Smad2)/c-jun signaling pathway, ultimately inducing neuronal injury. Our study provides evidence demonstrating that the overexpression of c-jun inhibits the protective role of MSCs-derived EV-miR-132 in neuronal injury. Upregulation of EV-derived miR-132 released from MSCs attenuates ischemic neuronal injury by inhibiting Smad2/c-jun pathways via the suppression of Acvr2b.

Inhibition of Cerebral Ischemia/Reperfusion Injury by MSCs-Derived Small Extracellular Vesicles in Rodent Models: A Systematic Review and Meta-Analysis

“…Only male animals were employed in 19 researches [26-31, 34, 35, 38-48]; other studies [32,33,36,37,49] did not report the sex of the animals. Anesthesia: 9 studies [26,29,30,34,35,37,38,41,47] used pentobarbital; 3 studies [28,31,48] used chloral hydrate; 1 study [36] used chloral hydrate and xylazine; 5 studies [27,39,40,43,46] used isoflurane; 1 study [32] used ketamine; 1 study [44] used ether; 1 study [45] used isoflurane oxygen/nitrous oxide mixture; 1 study [49] used uratan; and 2 studies [33,42] [26,33,38,39,46,48,49]; by gradient centrifuge method (2 studies) [35,43]; or by adherence method (2 studies) [30,36]; or by centrifugation in combination with filtration methods (1 study) [40]; or by filtration methods (1 study) [28]. 11 studies [27,29,…”

“…16 studies (67%) characterized MSCs for MSCs positive markers [26, 28, 32-38, 41-46, 49]. MSCs were fixed and stained with Alizarin Red S for osteogenic differentiation and/or Oil Red O for adipogenic differentiation in 8 studies [28,32,33,38,41,42,44,45] (Table 1). EVs were isolated via ultracentrifugation (UC) alone (12 studies) [29, 31, 34, 35, 37, 38, 41-44, 46, 49]; by UC in combination with filtration methods and/or isolation kits (4 studies) [30,32,40,47]; or by isolation kits (1 study) [45]; or by isolation kits in combination with low-speed centrifugation steps (1 study) [27]; or by sequential centrifugation (1 study) [36]; or by centrifugation (4 studies) [26,28,33,48].…”

From cerebral ischemia towards myocardial, renal, and hepatic ischemia: Exosomal miRNAs as a general concept of intercellular communication in ischemia-reperfusion injury

“…A great deal of miRNAs enveloped into MSC exosomes, such as miR-1-3p, miR-22-3p, miR-25, miR-26a, miR-26b-5p, miR-31, miR-126, miR-132, miR-138-5p, miR-146a-5p, miR-206, miR-223-3p, and miR-542-3p, were demonstrated to improve neuronal, astrocytic, oligodendrocytic, and microglial survival by downregulating target genes including KDM6B, p53, KLF9, CH25H, TRAF6, ACVR2B, LCN2, TLR4, RMRP, or CysLT2R. 38 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 Autophagy is also associated with the promotion of cell death via leading to cellular accumulation of toxic metabolites or cellular self-degradation. 88 Exosomal transfer of miRNAs from MSCs can reduce cell death by inhibiting excessive autophagy.…”

“…More interaction effects and related molecular targets under ischemic stroke are summarized in Table 1 and Figure 3 . 38 , 63 , 65 , 67 , 69 , 70 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 89 , 90 , 91 , 92 , 93 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 …”

“…94 More interaction effects and related molecular targets under ischemic stroke are summarized in Table 1and Figure 3. 38,63,65,67,69,70,[74][75][76][77][78][79][80][81][82][83][84][85][86][87][89][90][91][92][93][95][96][97][98][99][100][101][102]…”