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.

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.

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

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.

Antibodies to Plasmodium vivax reticulocyte binding protein 2b are associated with protection against P. vivax malaria in populations living in low malaria transmission regions of Brazil and Thailand.

BACKGROUND: The Plasmodium vivax Reticulocyte Binding Protein (PvRBP) family is involved in red blood cell recognition and members of this family are potential targets for antibodies that may block P. vivax invasion. To date, the acquisition of immunity against PvRBPs in low malaria transmission settings and in a broad age group of exposed individuals has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS: Total IgG antibody levels to six members of the PvRBP family (PvRBP1a, PvRBP1b, PvRBP2a, PvRBP2b, a non-binding fragment of PvRBP2c (PvRBP2cNB) and PvRBP2-P2) were measured in samples collected from individuals living in two regions of low P. vivax endemicity in Brazil and Thailand. In both settings, levels of total IgG to PvRBP1a, PvRBP2b, PvRBP2cNB, and PvRBP2P-2 increased significantly with age (rho = 0.17-0.49; P<0.001). IgG responses to PvRBP1a, PvRBP2b and PvRBP2cNB were significantly higher in infected individuals by using Wilcoxon's signed-rank test (P<0.001). Of the six PvRBPs examined, only antibodies to PvRBP2b were associated with protection against clinical malaria in both settings. CONCLUSION/SIGNIFICANCE: Our results indicate that PvRBP2b warrants further preclinical development as a blood-stage vaccine candidate against P. vivax. Total IgG responses to PvRBPs were also shown to be promising immunological markers of exposure to P. vivax infection.

Brominated polyunsaturated lipids from the Chinese sponge Xestospongia testudinaria as a new class of pancreatic lipase inhibitors.

Chemical analysis of the Chinese marine sponge Xestospongia testudinaria afforded a library of brominated polyunsaturated lipids including eight new compounds, named xestonarienes A-H (3-10) and thirteen known analogues (11-23). The structures of the new compounds were elucidated by detailed spectroscopic analysis and by comparison with literature data. The isolated lipids were evaluated for their inhibitory activity against pancreatic lipase (PL), an essential enzyme for efficient fat digestion and the major metabolite, 14, exhibited a marked inhibitory activity (IC50 = 3.11 µM), similar to that of the positive control Orlistat (IC50 = 0.78 µM). The preliminary structure-activity relationships on the series of compounds clearly evidenced that a terminal (E)-enyne functionality, a diyne within the chain, and methyl ester group are all key functional groups for the activity of this class of PL inhibitors. Further biological investigation on compound 14 revealed a significant decrease in the plasma triglyceride level following an oral lipid challenge in C57BLKS/J male mice. Acute toxicology study demonstrated that compound 14 was non-toxic up to 1600 mg/kg p.o in mice. This is the first report of the PL inhibitory activity for brominated polyunsaturated lipids and the obtained results qualify compound 14 as a potent and bioavailable drug candidate for a mild and safe treatment to prevent and reduce obesity.