miR-499 released during myocardial infarction causes endothelial injury by targeting α7-nAchR.

The surged systemic vascular inflammation after acute myocardial infarction (AMI) aggravates the atherosclerotic endothelial injury. To explore roles of miR-499 released from cardiomyocytes during AMI in endothelial injury. Using qPCR and ELISA, we discovered that patients with AMI had significantly increased plasma miR-499, which was directly correlated with serum thrombomodulin, a marker for endothelial injury. Plasma of AMI patients, when incubated with human umbilical vein endothelial cells (HUVECs), significantly increased the expression of endothelial injury markers, which could be abrogated by antagomiR-499. In vitro, neonatal rat cardiomyocytes subjected to hypoxia/reoxygenation (HX/R) released miR-499 that could be internalized into rat pulmonary microvascular endothelial cells (RPMECs), worsening the high glucose-induced injury. In silico analysis demonstrated that CHRNA7 encoding α7-nAchR is a target of miR-499, which was validated in cell lines expressing endogenous α7-nAchR. In high glucose-induced RPMECs injury model, miR-499 aggravated, whereas forced CHRNA7 expression ameliorated the injury. Moreover, the perfusate from Langendorff perfused rat heart subjected to HX/R contained higher level of miR-499 that significantly impaired the Bradykinin-mediated endothelium-dependent relaxation in both conduit and resistance arteries, which could be partially abrogated by antagomiR-499. Finally, the correlation between plasma miR-499 and endothelial injury was further confirmed in another cohort of AMI patients. We conclude that miR-499 released from injured cardiomyocytes contributes to the endothelial injury by targeting α7-nAchR. This study implies that miR-499 may serve as a potential target for the treatment of the surged vascular inflammation post-AMI.

[Role of over-expression of TBX3 and TBX18 in the enrichment and differentiation of human induced pluripotent stem cells into sinoatrial node-like cells].

OBJECTIVE: To explore the role of over-expression of TBX3 and TBX18 in inducing human induced pluripotent stem cells (HiPS) to enrich and differentiate into sinoatrial node-like cells. METHODS: The expression of stemness markers OCT3/4, SOX2, and NANOG in HiPS was detected by real-time fluorescence quantitative PCR (qRT- PCR), and compared with human embryonic stem cells (hESCs). Immunofluorescence staining was used to observe the expression of HiPS stemness markers OCT3/4, NANOG, SSEA4, and TRA-1-60. The HiPS were directional differentiated into cardiomyocytes, the expressions of ISL1, NK2 homeobox 5 (NKX2-5), ACTN1, and TNNT2 were detected by qRT-PCR, and human adult cardiomyocytes (hACM) were used as positive control. Immunofluorescence staining was used to observe the expressions of NKX2-5, cardiac troponin (cTnT), α-actinin, atria myosin light chain 2A (MLC-2A), and ventricular myosin light chain 2V (MLC-2V). The positive rate of α-actinin was detected by flow cytometry. On the 3rd day after HiPS were differentiated into cardiomyocytes (mesodermal stage), lentiviral over-expressions of sinoatrial node-related genes TBX3 and TBX18 were carried out for 21 days. The relative expressions of specific markers TBX3, TBX18, SHOX2, NKX2-5, HCN4, and HCN1 in sinoatrial node cells were detected by qRT-PCR, and compared with enhanced green fluorescent protein blank virus. RESULTS: OCT3/4, SOX2, and NANOG were highly expressed in HiPS and ESCs, and there was no significant difference in the relative expression of each gene ( P>0.05); OCT3/4 and NANOG were specifically distributed in the nucleus of HiPS, while SSEA4 and TRA-1-60 were distributed in the cell membrane. The relative expressions of ISL1 gene at 5, 7, 21, and 28 days and NKX2-5 gene at 7, 21, and 28 days of HiPS differentiation into cardiomyocytes were significantly higher than those of hACM ( P<0.05), and the relative expressions of ACTN1 and TNNT2 genes at 3, 5, 7, and 21 days of HiPS differentiation into cardiomyocytes were significantly lower than those of hACM ( P<0.05). NKX2-5 was expressed in most of the nuclei, cTnT and α-actinin, MLC-2A and MLC-2V signals were localized in the cytoplasm, presenting a texture-like structure of muscle nodules. Flow cytometry results showed that HiPS was successfully induced to differentiate into cardiomyocytes. The expressions of TBX18, SHOX2, HCN4, and HCN1 in the over-expression TBX3 group were up-regulated when compared with the control group, and difference in the relative expression of SHOX2 gene was significant ( P<0.05); the relative expression of NKX2-5 gene was lower than that in the control group, but there was no significant difference ( P>0.05). There was no significant difference in the relative expression of each gene between the over-expressed TBX18 group and the control group ( P>0.05). CONCLUSION: HiPS and hESCs have similar pluripotency, and we have established a stable method for maintaining and culturing the stemness of HiPS. A technological platform for the efficient differentiation of HiPS into cardiomyocytes has been successfully established. Although TBX3 and TBX18 do not play a significant role in promoting the enrichment and differentiation of HiPS into sinoatrial node-like cells, TBX3 shows a certain promoting trend, which can be further explored in the future.

Tetrahydrocurcumin Provides Neuroprotection in Experimental Traumatic Brain Injury and the Nrf2 Signaling Pathway as a Potential Mechanism.

The protective effect of tetrahydrocurcumin (THC) after experimental traumatic brain injury (TBI) has been demonstrated, as demonstrated by the inhibition of oxidative stress, mitochondrial dysfunction, and apoptosis. However, the mechanisms underlying this effect are still not well understood. This study was to investigate the neuroprotective effects of THC, and its potential mechanisms, in a rat model of TBI. To this end, rats were divided into 4 groups: the sham group, the TBI group, the TBI + vehicle (V) group, and the TBI + THC group. THC or V was administered via intraperitoneal injection to rats in the TBI + V and TBI + THC groups 30 min after TBI. After euthanasia (24 h after TBI), neurological scores, brain water content, and neuronal cell death in the cerebral cortex were recorded. Brain samples were collected after neurological scoring for further analysis. THC treatment alleviated brain edema, attenuated TBI-induced neuronal cell apoptosis, and improved neurobehavioral function. In addition, NFE2-related factor 2 (Nrf2) expression was upregulated following TBI. These results suggest that THC improves neurological outcome after TBI, possibly by activating the Nrf2 signaling pathway.