The long noncoding RNA expression profiles of paroxysmal atrial fibrillation identified by microarray analysis.

BACKGROUND: Long noncoding RNAs (lncRNAs) represent a novel class of noncoding RNAs that are involved in a variety of biological processes and human diseases. Recent evidence suggested that lncRNAs were associated with cardiac disorders. However, the roles of lncRNAs in paroxysmal atrial fibrillation (PAF) remain elusive. The purpose of the present study was to identify differentially expressed lncRNAs in PAF and predict their potential functions. METHODS: Between May 2014 and December 2015, a total of 67 patients, including 34 patients with PAF and 33 patients without PAF were recruited in this study. Of these participants, 3 PAF patients and 3 controls were used for the microarray analysis and a separate cohort (31 PAF patients and 30 controls) were used for further validation. LncRNA profiles in the leukocytes were detected by microarray. RESULTS: A total of 2095 and 1584 differentially expressed lncRNAs and mRNAs, respectively, were identified between the PAF patients and controls. Four lncRNAs (uc002nvy.3, ENST00000561094, uc004aef.3, ENST00000559960) were randomly selected for quantitative real-time PCR (qRT-PCR) in a separate cohort, validating that ENST00000559960 was upregulated and uc004aef.3 was downregulated in the PAF patients. uc002nvy.3 and ENST00000561094 showed no significant difference between PAF and the controls. Multiple logistic analyses showed that ENST00000559960 (OR 1.47; 95% CI 1.09 to 2.00; P=0.01) and uc004aef.3 (OR 0.63; 95% CI 0.41 to 0.96; P=0.03) were independently associated with PAF. Receiver operating characteristic (ROC) curves analyses revealed that ENST00000559960 and uc004aef.3 were modest predictors of PAF. The area under the curve (AUC) was 0.67±0.07 (95% CI 0.54-0.81; P=0.02) for uc004aef.3 and 0.70±0.07 (95% CI 0.56-0.83; P<0.01) for ENST00000559960. Bioinformatic analyses (lncRNAs classification and subgroup, gene ontology analysis, pathway analysis and gene co-expression network construction) were performed for predicting the role of lncRNAs. CONCLUSIONS: Our results demonstrated that lncRNA profiles were differentially expressed in the PAF leukocytes, and two lncRNAs (ENST00000559960 and uc004aef.3) may help in prediction of PAF. This motivates further investigation of the role of lncRNAs for PAF.

The Values of Coronary Circulating miRNAs in Patients with Atrial Fibrillation.

The mechanism of miRNA regulation in atrial fibrillation (AF) occurrence and development is still unclear, especially, the regulating values of coronary circulating miRNAs has not been reported. Based on our AF radiofrequency ablation clinical practice and previous miRNA study, we proposed a hypothesis that the coronary circulating miRNA might much better reflect the regulating state and metabolic level of myocardial miRNA in AF patient. To investigate the regulating values of coronary circulation miRNA, 90 AF patients were selected and compared with 90 healthy subjects, the changes of coronary circulating miRNA differential expression profile in the whole genome were observed in this study. We found out that compared with autologous peripheral blood (PB), 6 miRNAs were upregulated and 8 miRNA downregulated in AF patients' coronary sinus blood (CSB) significantly, especially, the expression of miR-1266, miR-4279 and miR-4666a-3p were obviously increased. Compared with normal donors' peripheral blood, 16 miRNAs were upregulated and 24 miRNAs downregulated dramatically in patients' peripheral blood, among them, the miR-3171 decreased, but miR-892a and miR-3149 increased significantly from the early to end stages of AF. Our results indicated that the coronary circulating miRNA can really reflect the regulating values of miRNA in AF patient; the level of miRNA change in 3 types of AF may reflect the severity of AF clinical and pathophysiological advance; The miR-892a, miR-3171 and miR-3149 may be used as biomarkers for earlier diagnosis, while miR-1266, miR-4279 and miR-4666a-3p may serve as potential intervening targets for AF patient in future.

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.

Effectiveness of Biodegradable Magnesium Alloy Stents in Coronary Artery and Femoral Artery.

OBJECTIVES: To access the biocompatibility, effectiveness, and safety of biodegradable magnesium (Mg) alloy stent (BMAS) in the coronary artery and femoral artery. BACKGROUND: Atherosclerosis is a lesion of cardiovascular system, including the diseases in heart and blood vessels. METHODS: The aluminum (Al) and zinc (Zn)-based BMAS was designed by cold drawing methods. Forty healthy immunized mongrel dogs were randomly divided into 8 groups. Five dogs who have not been treated with stent were included in control group. The other dogs were implanted with an absorbable magnesium (Mg) alloy in the coronary and/or femoral artery, and their artery angiography were observed at 7 time points (1, 3, 5, 7, 14, 21, and 28 days; n = 5) follow-up. Dogs from each cohort were sacrificed following angiography for pathology assessment. The histological response including inflammatory response, thrombosis, and intimal hyperplasia were analyzed by hematoxylin-eosin staining. Lumen area (La), intimal hyperplasia area (IHa), and the ratio of IHa were calculated by image analysis software. RESULTS: The thin-walled BMAS were designed and produced by cold-drawing technology. Fifty-one devices were implanted into coronary artery of 35 dogs successfully. During the follow-up days, the angiography of coronary artery and femoral artery had confirmed that the lumen was clear and there were no elastic recoil and thrombosis. The stents were completely disappeared at 7 days after implantation. Moderate intimal hyperplasia was found at 14 days after implantation. CONCLUSION: The BMAS stent proved to be of good biocompatibility, safety, and effectiveness. (J Interven Cardiol 2015;XXXX:XX-XX).

[Radiofrequency ablation can reverse the abnormal circulating microRNA expression changes in patients with atrial fibrillation].

OBJECTIVE: To observe the change of circulating microRNAs(miRNAs), regulatory mechanism in patients with atrial fibrillation (AF) before and after radiofrequency ablation (RFA). METHODS: From January 2011 to December 2013, peripheral blood samples were taken from 30 AF patients (10 paroxysmal, 10 persistent and 10 permanent AF) before and 3 months after RFA. The total RNA was extracted and hybridized with the miRNA chips, and the differential expression of miRNA and clustering analysis in whole genome were made with Volcano Plot and tMEV software respectively, and validated by real-time PCR. The target gene analysis of miRNAs was predicted through the Mirbase, Miranda and Targetscan databases. Results were compared with those from 10 healthy subjects (control group). RESULTS: Compared with control group, the expressions of 25 miRNAs were down-regulated before RFA and up-regulated after RFA in AF group, while other 40 miRNAs expression changed in the opposite way; among them, the expressions of 7 miRNAs including miR-199a-3p/miR-199b-3p were down- regulated >1.5-fold before RFA and up-regulated>100-fold after RFA; oppositely, 6 miRNAs including miR-BART8-3p were up-regulated>1.5-fold before RFA and down-regulated>10-fold after RFA. Interestingly, 6 miRNAs including miR-30b-5p, which were involved in AF-related electrical and structural remodeling, were down-regulated>5-fold before RFA, but up-regulated>50-fold after RFA. Four miRNAs including miR-377-5p, which were involved in the regulation of CACNA1C ICaL channel protein, were different before and after RFA. CONCLUSION: miRNAs regulate the occurrence and development of AF. RFA can change the expression of miRNAs in AF patients, which may be important for reversing the electrical and structural remodeling and maintaining sinus rhythm after RFA. miRNAs, such as miR-30b-5p, miR-377-5p and miR-199a-3p/miR-199b-3p etc., might become the target markers for early diagnosis and intervention of AF in future.

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.