M2 Macrophage-Derived Exosomes Inhibit Apoptosis of HUVEC Cell through Regulating miR-221-3p Expression.

Atherosclerosis (AS) is associated with high morbidity and mortality rates and currently has no effective treatment. This study was aimed at investigating the role of macrophage exosomes in the inflammation and apoptosis after HUVEC injury. We established the HUVEC injury model using 100 mg/L oxidized low-density lipoprotein (ox-LDL) or 50 ng/mL tumor necrosis factor-α (TNF-α). Cell proliferation was assessed using cell counting kit-8 (CCK8) assays, and the expression of miR-221, TNF-α, and IL-6, IL-10, and IL-1ß was detected using quantitative real-time PCR (qRT-PCR). The apoptotic rate was analyzed by the TUNEL method, and the expressions of apoptosis-related proteins Bcl2, Caspase-3, and c-myc were detected by western blotting. Finally, miR-221-3p mimics and miR-221-3p inhibitors were constructed by liposome transfection to determine the mechanism of action of macrophage exosomes on HUVEC injury. The expression levels of IL-6, IL-1ß, and TNF-α in the injury groups were higher than those in the normal group, but the expression of IL-10 in the injury groups was lower than that in the normal group. Meanwhile, the apoptotic rate of the HUVEC cell injury group was higher than that of the normal group. In contrast, the expression levels of IL-6, IL-1ß, and TNF-α were lower in the M2 macrophage exosome (M2-Exo) group, but the expression of IL-10 was higher compared with the control group. The apoptosis rate was reduced in the M2-Exo group, and the expression of the proapoptotic gene Caspase-3 was reduced, while the expression of the antiapoptotic gene Bcl2 was increased. Liposome transfection of miR-221-3p mimics was able to enhance the effect of M2 macrophage exosomes. Thus, M2-Exo promotes HUVEC cell proliferation and inhibits HUVEC cell inflammation and apoptosis. miR-221-3p overexpression attenuates HUVEC cell injury-induced inflammatory response and apoptosis, while miR-221-3p gene inhibition enhances this inflammatory response and apoptosis.

HIV-1 Vpr potently induces programmed cell death in the CNS in vivo.

The human immunodeficiency virus type I (HIV-1) accessory protein Vpr has been associated with the induction of programmed cell death (apoptosis) and cell-cycle arrest. Studies have shown the apoptotic effect of Vpr on primary and established cell lines and on diverse tissues including the central nervous system (CNS) in vitro. However, the relevance of the effect of Vpr observed in vitro to HIV-1 neuropathogenesis in vivo, remains unknown. Due to the narrow host range of HIV-1 infection, no animal model is currently available. This has prompted us to consider a small animal model to evaluate the effects of Vpr on CNS in vivo through surrogate viruses expressing HIV-1Vpr. A single round of replication competent viral vectors, expressing Vpr, were used to investigate the apoptosis-inducing capabilities of HIV-1Vpr in vivo. Viral particles pseudotyped with VSV-G or N2c envelopes were generated from spleen necrosis virus (SNV) and HIV-1-based vectors to transduce CNS cells. The in vitro studies have demonstrated that Vpr generated by SNV vectors had less apoptotic effects on CNS cells compared with Vpr expressed by HIV-1 vectors. The in vivo study has suggested that viral particles, expressing Vpr generated by HIV-1-based vectors, when delivered through the ventricle, caused loss of neurons and dendritic processes in the cortical region. The apoptotic effect was extended beyond the cortical region and affected the hippocampus neurons, the lining of the choroids plexus, and the cerebellum. However, the effect of Vpr, when delivered through the cortex, showed neuronal damage only around the site of injection. Interestingly, the number of apoptotic neurons were significantly higher with HIV-1 vectors expressing Vpr than by the SNV vectors. This may be due to the differences in the proteins expressed by these viral vectors. These results suggest that Vpr induces apoptosis in CNS cells in vitro and in vivo. To our knowledge, this is the first study to investigate the apoptosis-inducing capabilities of HIV-1Vpr in vivo in neonatal mice. We propose that this, in expensive animal model, may be of value to design-targeted neuroprotective therapeutics.

Claudin-1 regulates pulmonary artery smooth muscle cell proliferation through the activation of ERK1/2.

Tumor necrosis factor alpha (TNF-α), a crucial inflammatory cytokine, is involved in the pathogenesis of pulmonary arterial hypertension (PAH). TNF-α can induce claudin-1 (CLDN1) expression and CLDN1 has been reported to be associated with the regulation of cellular functions including cell proliferation, migration. Thus, we aimed to explore whether CLDN1 participated in the etiology of PAH. Our study showed that CLDN1 expression was markedly increased in the lungs of rats with monocrotaline(MCT)-induced PAH, especially in the pulmonary arterial smooth muscle sections. We also found that CLDN1 expression in primary human PASMCs was up-regulated by TNF-α, and the Nuclear factor-κB (NF-κB) inhibitor BAY 11-7082 suppressed CLDN1 up-regulation by TNF-a. CLDN1 overexpression by adenoviral transduction promoted PASMCs proliferation, while knockdown of CLDN1 by siRNA inhibited TNF-α-induced cell proliferation. Mechanistic studies revealed that CLDN1 regulated human PASMC proliferation through the activation of ERK1/2. Together, our findings indicate that up-regulation of CLDN1 promotes PASMC proliferation contributing to pulmonary arterial remodeling in PAH.

[Role of Smac in cisplatin-induced apoptosis of non-small cell lung cancer cells].

OBJECTIVE: To investigate the role of Smac in cisplatin-induced apoptosis of non-small lung cancer cells in vitro. METHODS: Non-small cell lung cancer A549 cells were incubated in the presence of cisplatin at different concentrations, and the cell proliferation status was observed using MTT assay. Flow cytometry was used for evaluation of the apoptosis of the incubated cells, and the expressions of Smac mRNA and protein were detected by RT-PCR and Western blotting, respectively. RESULTS: Cisplatin inhibited the proliferation and induced apoptosis of A549 cells both in a concentration-dependent manner. Cisplatin also increased the expression of Smac at both the mRNA and protein levels, which was also concentration-dependent. CONCLUSION: Increased Smac expression may play a critical role in cisplatin-induced apoptosis of the non-small cell lung cancer cells in vitro.

Cryopreservation of primary human hepatocytes: the benefit of trehalose as an additional cryoprotective agent.

Problems with the limited availability of human hepatocytes for cell transplantation may be overcome by efficient cryopreservation techniques and formation of appropriate cell banking. In this study we investigated the effect of the disaccharide trehalose on the cryopreservation of human hepatocytes. For analysis, liver cells were frozen in culture medium containing 10% dimethyl sulfoxide (DMSO) that was supplemented with varying concentrations of trehalose. During the postthawing culture period, viability, plating efficiency, total protein, cell proliferation, enzyme leakage, albumin and urea formation, as well as phase I and II metabolism were analyzed. In the pilot study, among the concentrations investigated, 0.2 M trehalose showed the best overall outcome. Compared to the use of DMSO alone, we found significant improvement in postthaw cell viability (62.9 +/- 13 vs. 46.9 +/- 11%, P < 0.01) and plating efficiency (41.5 +/- 18 vs. 17.6 +/- 13%, P < 0.01) in the trehalose group. The use of trehalose as an additive for cryopreserving human hepatocytes resulted in a significantly increased total protein level in the attached cells, higher secretion of albumin and a lower aspartate aminotransferase (AST) level after thawing. In conclusion, the use of trehalose as cryoprotective agent significantly improves the outcome of human hepatocyte cryopreservation.