miR-666-3p Mediates the Protective Effects of Mesenchymal Stem Cell-derived Exosomes Against Oxygen-glucose Deprivation and Reoxygenation- induced Cell Injury in Brain Microvascular Endothelial Cells via Mitogen-activated Protein Kinase Pathway.

BACKGROUND: Previous studies have reported that mesenchymal stem cell (MSC)- derived exosomes can protect primary rat brain microvascular endothelial cells (BMECs) against oxygen-glucose deprivation and reoxygenation (OGD/R)-induced injury. OBJECTIVE: The aim was to identify the key factors mediating the protective effects of MSC-derived exosomes. METHODS: Primary rat BMECs were either pretreated or not pretreated with MSC-derived exosomes before exposure to OGD/R. Naïve cells were used as a control. After performing small RNA deep sequencing, quantitative reverse transcription polymerase chain reaction was performed to validate microRNA (miRNA) expression. The effects of rno-miR-666-3p on cell viability, apoptosis, and inflammation in OGD/R-exposed cells were assessed by performing the Cell Counting Kit 8 assay, flow cytometry, and enzyme-linked immunosorbent assay, respectively. Moreover, the role of rno-miR-666-3p in regulating gene expression in OGD/R-exposed cells was studied using mRNA deep sequencing. Lastly, to evaluate whether mitogen-activated protein kinase 1 (MAPK1) was the target of rno-miR-666-3p, western blotting and the dual-luciferase assay were performed. RESULTS: MSC-derived exosomes altered the miRNA expression patterns in OGD/R-exposed BMECs. In particular, the expression levels of rno-miR-666-3p, rno-miR-92a-2-5p, and rnomiR- 219a-2-3p decreased in OGD/R-exposed cells compared with those in the control; however, MSC-derived exosomes restored the expression levels of these miRNAs under OGD/R conditions. rno-miR-666-3p overexpression enhanced cell viability and alleviated the apoptosis of OGD/R-exposed cells. Moreover, rno-miR-666-3p suppressed OGD/R-induced inflammation. mRNA deep sequencing revealed that rno-miR-666-3p is closely associated with the MAPK signaling pathway. Western blotting and the dual-luciferase assay confirmed that MAPK1 is the target of rnomiR- 666-3p. CONCLUSION: MSC-derived exosomes restore rno-miR-666-3p expression in OGD/R-exposed BMECs. Moreover, this specific miRNA exerts protective effects against OGD/R by suppressing the MAPK signaling pathway.

Mesenchymal Stem Cell-derived Exosomes Rescue Oxygen-Glucose Deprivation-induced Injury in Endothelial Cells.

OBJECTIVE: The effects of mesenchymal stem cell (MSC)-derived exosomes on brain microvascular endothelial cells under oxygen-glucose deprivation (OGD), which mimic cells in deep hypothermic circulatory arrest (DHCA) in vitro, are yet to be studied. METHODS: MSCs were co-cultured with primary rat brain endothelial cells, which were then exposed to OGD. Cell viability, apoptosis, the inflammatory factors (IL-1ß, IL-6, and TNF-α), and the activation of inflammation-associated TLR4-mediated pyroptosis and the NF-κB signaling pathway were determined. Furthermore, exosomes derived from MSCs were isolated and incubated with endothelial cells to investigate whether the effect of MSCs is associated with MSCderived exosomes. Apoptosis, cell viability, and the inflammatory response were also analyzed in OGD-induced endothelial cells incubated with MSC-derived exosomes. RESULTS: OGD treatment promoted endothelial cell apoptosis, induced the release of inflammatory factors IL-1ß, IL-6, and TNF-α, and inhibited cell viability. Western blot analysis showed that OGD treatment-induced TLR4, and NF-κB p65 subunit phosphorylation and caspase-1 upregulation, while co-culture with MSCs could reduce the effect of OGD treatment on endothelial cells. As expected, the effect of MSC-derived exosomes on OGD-treated endothelial cells was similar to that of MSCs. MSC-derived exosomes alleviated the OGD-induced decrease in the viability of endothelial cells, and increased levels of apoptosis, inflammatory factors, and the activation of inflammatory and inflammatory focal pathways. CONCLUSION: Both MSCs and MSC-derived exosomes attenuated OGD-induced rat primary brain endothelial cell injury. These findings suggest that MSC-derived exosomes mediate at least some of the protective effects of MSCs on endothelial cells.

Efficacy and safety of low-dose clopidogrel after 12-month dual antiplatelet therapy for patients having drug-eluting stent implantation.

BACKGROUND: To prevent stent thrombosis (ST) after implantation of drug-eluting stents (DESs) in patients with coronary heart disease, 12-month dual antiplatelet therapy (DAPT) is recommended. However, the optimal long-term antiplatelet regimen is not clear for the patients who have completed the 12-month DAPT. METHODS: We reviewed the data of 755 consecutive patients who had undergone percutaneous coronary intervention (PCI) three years ago and completed 12-month DAPT. They were divided into three groups according to the antiplatelet medication they had used for two years after 12-month DAPT [low-dose clopidogrel (Talcom(®), 25mg/d), clopidogrel (Plavix(®), 75mg/d) and aspirin (100 mg/d)]. The efficacy (a composite incidence of cardiac death, myocardial infarction and target vessel revascularization) and safety (incidences of bleeding, gastrointestinal trouble and drug discontinuation) were compared among the three groups. RESULTS: The rates of multi-vessel lesions, prior MI, hemoglobin A1C (HbA1c) and low-density lipoprotein cholesterol were significantly higher in the clopidogrel (75 mg/day) group than in the other two groups (P>0.05 for both comparisons). There was no significant difference in the overall composite incidence of cardiac death, myocardial infarction and target vessel revascularization in the three groups at three years after PCI. The rates of bleeding (especially minor bleeding), gastrointestinal trouble, drug discontinuation and any blood transfusion were markedly lower in the low-dose clopidogrel (25 mg/d) group than in the other two treatment groups (P<0.05). CONCLUSIONS: The 25-mg maintenance dose of clopidogrel after 12-month DAPT may be more preferable to Chinese patients who have undergone DES implantation, because of its lower cost but no less efficacy and safety.

1,5-Diamino-tetra-zolium chloride.

The title compound, CH(5)N(6) (+)·Cl(-), crystallized with two indepedent 1,5-diamino-tetra-zolium cations and two independent chloride anions in the asymmetric unit. In the crystal, there are a number of N-H⋯Cl hydrogen-bonding inter-actions, which generate a three-dimensional network.

(5Z,7Z)-6,8-Dimethyl-9H-tetra-zolo[1,5-b][1,2,4]triazepine.

The mol-ecule of the title compound, C(6)H(8)N(6), is approximately planar, with a maximum deviation from planarity of 0.099 (1) Å. In the crystal, mol-ecules are linked to each other via pairs of N-H⋯N hydrogen bonding, forming inversion dimers. The crystal structure is further stabilized by π-π stacking inter-actions, with a centroid-centroid distance of 3.419 (1) Å.

N-(5-Amino-1H-tetra-zol-1-yl)formamide.

In the title compound, C(2)H(4)N(6)O, the planar [maximum deviation = 0.006 (2) Å] amino-tetra-zole group makes a dihedral angle of 83.65 (8)° with the formamide unit. In the crystal structure, inter-molecular N-H⋯N, N-H⋯O and C-H⋯N hydrogen bonds are responsible for the formation of a three-dimensional network.

1-Isopropyl-ideneamino-1H-tetra-zol-5-amine.

The mol-ecule of the title compound, C(4)H(8)N(6), assumes an approximately planar structure, the methyl C atoms and the C atom to which they are bonded being out of the mean tetrazole ring plane by 0.108 and 0.139, and 0.144 Å, respectively. π-π stacking between parallel tetra-zole rings [centroid-centroid distance = 3.4663 (11) Å] is observed in the crystal structure. Inter-molecular N-H⋯N hydrogen bonding further helps to stabilize the crystal structure.

N-(1-Diacetyl-amino-1H-tetra-zol-5-yl)acetamide.

In the crystal structure of the title compound, C(7)H(10)N(6)O(3), there are N-H⋯O, N-H⋯N and C-H⋯O inter-actions, generating a three-dimensional supra-molecular network structure. A short intermolecular O⋯C contact of 2.8994 (18) Šis alsopresent in the crystal structure, but no π-π contacts are observed.