Enrichment of miR-17-5p enhances the protective effects of EPC-EXs on vascular and skeletal muscle injury in a diabetic hind limb ischemia model

Abstract

Background/aims Diabetes mellitus (DM) is highly susceptible to diabetic hind limb ischemia (DHI). MicroRNA (MiR)-17-5p is downregulated in DM and plays a key role in vascular protection. Endothelial progenitor cell (EPC)-released exosomes (EPC-EXs) contribute to vascular protection and ischemic tissue repair by transferring their contained miRs to target cells. Here, we investigated whether miR-17-5p-enriched EPC-EXs (EPC-EXsmiR-17-5p) had conspicuous effects on protecting vascular and skeletal muscle in DHI in vitro and in vivo. Methods EPCs transfected with scrambled control or miR-17-5p mimics were used to generate EPC-EXs and EPC-EXsmiR-17-5p. Db/db mice were subjected to hind limb ischemia. After the surgery, EPC-EXs and EPC-EXsmiR-17-5p were injected into the gastrocnemius muscle of the hind limb once every 7 days for 3 weeks. Blood flow, microvessel density, capillary angiogenesis, gastrocnemius muscle weight, structure integrity, and apoptosis in the hind limb were assessed. Vascular endothelial cells (ECs) and myoblast cells (C2C12 cells) were subjected to hypoxia plus high glucose (HG) and cocultured with EPC-EXs and EPC-EXsmiR-17-5p. A bioinformatics assay was used to analyze the potential target gene of miR-17-5p, the levels of SPRED1, PI3K, phosphorylated Akt, cleaved caspase-9 and cleaved caspase-3 were measured, and a PI3K inhibitor (LY294002) was used for pathway analysis. Results In the DHI mouse model, miR-17-5p was markedly decreased in hind limb vessels and muscle tissues, and infusion of EPC-EXsmiR-17-5p was more effective than EPC-EXs in increasing miR-17-5p levels, blood flow, microvessel density, and capillary angiogenesis, as well as in promoting muscle weight, force production and structural integrity while reducing apoptosis in gastrocnemius muscle. In Hypoxia plus HG-injured ECs and C2C12 cells, we found that EPC-EXsmiR-17-5p could deliver their carried miR-17-5p into target ECs and C2C12 cells and subsequently downregulate the target protein SPRED1 while increasing the levels of PI3K and phosphorylated Akt. EPC-EXsmiR-17-5p were more effective than EPC-EXs in decreasing apoptosis and necrosis while increasing viability, migration, and tube formation in Hypoxia plus HG-injured ECs and in decreasing apoptosis while increasing viability and myotube formation in C2C12 cells. These effects of EPC-EXsmiR-17-5p could be abolished by a PI3K inhibitor (LY294002). Conclusion Our results suggest that miR-17-5p promotes the beneficial effects of EPC-EXs on DHI by protecting vascular ECs and muscle cell functions.

Diagnostic Role of Cell-Free miRNAs in Identifying Placenta Accreta Spectrum during First-Trimester Screening

“…The anti-inflammatory effect and promotion of the angiogenesis in the skeletal muscle injury model were found for miR-320a and miR-26a via reduction of the protein expression of their target genes-PTEN and TLR3, respectively [57]. Enriched by miR-17-5p exosomes from endothelial progenitor cell decrease cell apoptosis, increase microvessel density and capillary angiogenesis as well as promote muscle structural integrity in a diabetic hind-limb ischemia mode through increasing the levels of PI3K and phosphorylated Akt [58]. The participation of miR-92a-3p in exosome-mediated angiogenesis was found in retinoblastoma by targeting transcription factor KLF2 [59] which is able to modulate tumor proliferation and metastasis [60].…”

Systemic delivery of murine SOD2 mRNA to experimental abdominal aortic aneurysm mitigates expansion and rupture

“…Thus, the high detection of Plex (pleckstrin), Trpc6 (transient receptor potential channel 6), Rasa3 (Ras p21 protein activator 3), and Alox12 (arachidonate 12-lipoxygenase) may portend risk of arterial aneurysm rupture while overexpression of SOD2 consistently downregulated these pathways, potentially protecting the animals from aneurysm rupture and sudden death. Subsequent analysis of significantly enriched pathways derived from AAA proteomes revealed that HA-SOD2 mRNA NP treatment modulated cellular response to stress, potentially through the regulation of microRNA (miR)17-5p, which inhibits apoptosis, 23,24 and miR 181a-5p, which regulates vascular inflammation/endothelial cell senescence via STAT3 signaling pathway (Figure 7C-D). 25,26 Furthermore, HA-SOD2 mRNA NP treatment inhibited pathways involved in platelet activation/aggregation (Csk, Lyn, Arrb1, Plek), cell adhesion (Isg15, Lyn), and MAPK1/3 signaling (Rasa3, Csk, Arrb1) (Figure 7C-D).…”