N-ethylmaleimide­sensitive factor siRNA inhibits the release of Weibel-Palade bodies in endothelial cells.

The aim of the present study was to examine the effect of small interfering RNA (siRNA) methods on the expression of N­ethylmaleimide sensitive factor (NSF) and Weibel­Palade body (WPB) release in endothelial cells. A small hairpin RNA (shRNA), mediated with an adenovirus vector, was designed to target the N­terminal functional area of NSF. Subsequently, viruses were transfected into human aortic endothelial cells. The mRNA and protein expression levels of NSF were detected using reverse transcription­quantitative polymerase chain reaction and Western blot analyses, respectively, and the release of WPBs in the endothelial cells was examined using immunofluorescence. The mRNA expression of NSF in the endothelial cells, which were transfected with the adenoviruses carrying the NSF­shRNA was significantly decreased, compared with the negative control group (P=0.035) and blank control group (P=0.02). In addition, the mRNA expression of NSF was gradually decreased as duration increased; there were marked differences between the 24, 48 and 72 h groups (P<0.05). The protein expression of NSF was significantly decreased in the experimental group, compared with the negative control group (P=0.004) and blank control group (P=0.031), however, no difference was observed between the negative control and blank control groups (P=0.249). The immunofluorescence staining showed that the release of WPBs in the endothelial cells induced with thrombin was inhibited markedly following transfection with the virus carrying the NSF­shRNA. Therefore NSF­siRNA inhibited the mRNA and protein expression levels of NSF, and inhibited the release of WPBs in endothelial cells induced with thrombin. These results suggested that NSF-siRNA may be valuable for preventing and treating atherosclerosis and acute coronary syndrome.

Safety of umbilical cord blood-derived mesenchymal stem cells (MSCs) following 5-azaserine induction and inhibition of human cardiac myocyte apoptosis by MSCs.

OBJECTIVE: To further study the safety and effect of the umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) on apoptosis of human cardiac myocyte (HCM). METHODS: The UCB was collected at the time of delivery with informed consent obtained from 10 donors. The UCB-derived MSCs was treated with 5-azaserine (5-AZA), and further introduced differentiation into cardiomyocytes. The telomerase activity, G-banding patterns of chromosomal karyotypes, tumor formation in nude mice, reverse transcription polymerase chain reaction (RT-PCR), and the inhibited apoptosis of UCB-derived MSCs were further investigated. This study was carried out in the laboratory of Beijing Shijitan Hospital, Beijing, China and Inheritance Research Section of Chinese Medical Institute, Beijing, China from July 2005 to December 2007. RESULTS: The MSCs-derived from UCB were differentiated into cardiomyocytes in vitro, possessed telomerase activity after 5-AZA induction, and no abnormal chromosomal karyotypes were observed. Expression of p53, cyclinA, cdk2, -actin, C-fos, h-TERT and c-myc were similar in MSCs before and after 5-AZA treatment. There was no tumor formation injected into nude mice. The UCB-derived MSCs significantly inhibited apoptosis of human cardiomyocytes. CONCLUSION: Umbilical cord blood-derived MSCs are safe and effective source of cell-transplantation treatment, and can inhibit the apoptosis of human cardiomyocytes in co-cultured.

Rac1 regulates the release of Weibel-Palade Bodies in human aortic endothelial cells.

BACKGROUND: The release of Weibel-Palade Bodies (WPB) is a form of endothelial cell activation. But the signal transduction pathway leading to WPB release is not yet defined. We hypothesized that small G-protein rac1 and reactive oxygen species (ROS) mediate the ligand induced release of Weibel-Palade Bodies. METHODS: We tested this hypothesis by using wild-type and mutant adenoviral rac1 expression vectors, and by manipulating the production and destruction of superoxide and hydrogen peroxide in human aortic endothelial cells (HAEC). RESULTS: Thrombin (1.0 Unit, 30 min) induced the increase of WPB release by 3.7-fold in HAEC, and that H2O2 (0.1 mmol/L, 30 min) induced by 4.5-fold. These results correlated with thrombin-stimulated activation of rac-GTP binding activity by 3.5-fold, and increase of ROS production by 3.4-fold. The dominant negative adenoviral rac-N17 gene transfer dramatically inhibited the release of WPB by 64.2% (control) and 77.3% (thrombin-stimulation), and decreased ROS production by 65.5% (control) and 83.6% (thrombin-stimulation) compared with non-infected cells, respectively. Anti-oxidants, catalase and N-acetyl-cysteine significantly decreased the release of WPB by 34% and 79% in control cells, and further decreased by 63.6% and 46.7% in rac-N17 transferred cells compared with non-infected cells. We also confirmed that rac1 was located upstream of ROS in the WPB release pathway. CONCLUSIONS: Small G-protein rac1 medicates ligand-induced release of Weibel-Palade Bodies in human aortic endothelial cells, and the signal pathway of WPB release is a rac1-dependent ROS regulating mechanism.

Nitric oxide regulates exocytosis by S-nitrosylation of N-ethylmaleimide-sensitive factor.

Nitric oxide (NO) inhibits vascular inflammation, but the molecular basis for its anti-inflammatory properties is unknown. We show that NO inhibits exocytosis of Weibel-Palade bodies, endothelial granules that mediate vascular inflammation and thrombosis, by regulating the activity of N-ethylmaleimide-sensitive factor (NSF). NO inhibits NSF disassembly of soluble NSF attachment protein receptor (SNARE) complexes by nitrosylating critical cysteine residues of NSF. NO may regulate exocytosis in a variety of physiological processes, including vascular inflammation, neurotransmission, thrombosis, and cytotoxic T lymphocyte cell killing.