LncRNA HOXA-AS3 Promotes the Progression of Pulmonary Arterial Hypertension through Mediation of miR-675-3p/PDE5A Axis.

Pulmonary arterial hypertension (PAH) seriously threatens the elder people. Long non-coding RNAs (lncRNAs) are involved in multiple diseases. However, the study of the lncRNAs in the occurrence of PAH is just beginning. For this, we sought to explore the biological function of lncRNA HOXA cluster antisense RNA 3 (HOXA-AS3) in PAH. Hypoxia (HYP) was used to mimic in vitro model of PAH. Gene and protein expressions in cells were detected by q-PCR and Western blotting, respectively. In addition, cell proliferation and viability were tested by CCK-8 and MTT assay. Cell apoptosis was measured by flow cytometry. Wound healing was used to detect cell migration. Furthermore, the connection of HOXA-AS3, miR-675-3p, and phosphodiesterase 5A (PDE5A) was verified by dual-luciferase report assay. HOXA-AS3 and PDE5A were upregulated in human pulmonary artery smooth muscle cells (HPASMCs) in the presence of HYP, while miR-675-3p was downregulated. Moreover, knockdown of HOXA-AS3 suppressed the growth and migration of HPASMCs, but induced the apoptosis. Overexpression of miR-675-3p achieved the same effect. MiR-675-3p inhibitor or overexpression of PDE5A notably reversed the inhibitory effect of HOXA-AS3 knockdown on PAH. Finally, HOXA-AS3 could sponge miR-675-3p, and PDE5A was directly targeted by miR-675-3p. HOXA-AS3 increased the development of PAH via regulation of miR-675-3p/PDE5 axis, which could be the potential biomarker for treatment of PAH.

MicroRNA-495 Ameliorates Cardiac Microvascular Endothelial Cell Injury and Inflammatory Reaction by Suppressing the NLRP3 Inflammasome Signaling Pathway.

BACKGROUND/AIMS: In recent years, microRNA-495 (miR-495) has been reported to be a tumor-suppressor miR that is down-modulated in cancers. However, its potential mechanism remains unknown. Therefore, this study aimed to demonstrate the role of miR-495 in cardiac microvascular endothelial cell (CMEC) injury and inflammatory reaction by mediating the pyrin domain-containing 3 (NLRP3) inflammasome signaling pathway. METHODS: Overall, 40 mice were assigned into myocardial ischemia/reperfusion injury (MIR) and sham groups. After model establishment, the levels of troponin T (TnT), troponin I (TnI), N-terminal pro-B-type natriuretic peptide (NT-proBNP), creatine kinase isoenzyme MB (CK-MB), myoglobin (MYO), tumor necrosis factor-alpha (TNF-α), and interleukin 1beta (IL-1ß) were detected by Enzyme-Linked Immunosorbent Assay (ELISA). Apoptosis was evaluated using Terminal deoxy (d)-UTP nick end labeling (TUNEL) staining, the level of NLRP3 protein was determined by immunohistochemical assay, and miR-495 was detected by in situ hybridization (ISH). The infarct size was determined using 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. The expression of miR-495 and the mRNA and protein levels of NLRP3, TNF-α, IL-1ß, IL-18 and caspase-1 were evaluated by RT-qPCR and western blot analysis. After transfection, the cells were treated with a miR-495 mimic, a miR-495 inhibitor, or siNLRP3. Cell proliferation was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and cell cycle and apoptosis by flow cytometry. RESULTS: Mice with myocardial I/R injury had elevated levels of TnT, TnI, NT-proBNP, CK-MB, MYO, TNF-α and IL-1ß; enhanced cell apoptosis; increased expression of NLRP3, TNF-α, IL-1ß, IL-18 and caspase-1; and decreased miR-495 expression. MiR-495 was confirmed to target NLRP3. Moreover, miR-495 reduced the mRNA and protein levels of NLRP3, TNF-α, IL-1ß, IL-18 and caspase-1, inhibited cell apoptosis and decreased cells at the G0/G1 phase while improving cell proliferation and increasing cells at the S phase. However, the effects of NLRP4 were proved to be reciprocal. CONCLUSION: In conclusion, the current study indicated that miR-495 improved CMEC injury and inflammation by suppressing the NLRP3 inflammasome signaling pathway.

[Impact of five genetic polymorphisms on inter-individual variation in warfarin maintenance dose].

OBJECTIVE: To investigate the effect of genetic polymorphisms in VKORC1, CYP2C9, GGCX, EPHX1, APOE genes on inter-individual variation in warfarin maintenance dose. METHODS: Two hundred and forty-nine patients with stable warfarin dose were enrolled in this study, and the clinical data and blood samples of the patients were collected. Genotypes for the 5 genes were determined by using PCR and denaturing high performance liquid chromatography (DHPLC) assay. The warfarin maintenance doses were compared among patients with different genotypes of the 5 genes, and a warfarin stable dosing algorithm was derived based on genetic and non-genetic factors. RESULTS: Of the 5 genes, VKORC1, CYP2C9 and GGCX were associated with warfarin stable dose. The multiple linear regression analysis indicated that VKORC1, CYP2C9 and GGCX genes, age and weight, had significant influence on inter-individual variation in warfarin stable dose, which contributed 30.2%, 22.8%, 1.5%, 4.7% and 6.7% respectively. The warfarin stable dosing algorithm acquired from the optimal regression model could explain 57.8% variation in warfarin dose. CONCLUSION: This study suggested that genetic factors are the major determinants of the warfarin maintenance dose, and warfarin stable dosing algorithm may be useful for helping clinicians to prescribe warfarin with greater safety and efficiency.

Influence of GGCX genotype on warfarin dose requirements in Chinese patients.

INTRODUCTION: It has been widely accepted that genetic factors were the major sources of the variation in warfarin dose. This study is intended to investigate whether the 3261G>A variation in GGCX gene influences stable warfarin dose in Chinese patient population. MATERIALS AND METHODS: A total of 217 patients with stable warfarin dose were enrolled. Genomic DNA was extracted from each subject and the genotype of GGCX 3261G>A was determined by using of denaturing high-performance liquid chromatography (DHPLC). Least significant difference tests (LSDs) were used to compare dose with genotypes. Analysis of variance (ANVOA) was used to calculate the proportion of warfarin dose that could be explained by variation in genotype. RESULTS: In the total of 217 subjects, 84 patients (38.7%) were GG homozygote, whereas 117 (53.9%) were GA heterozygote and 16 (7.4%) were AA homozygote. Patients with the GGCX 3261AA genotype had a significantly higher average daily maintenance dose (3.39 ± 1.40 mg) than those with the GG genotype (2.69 ± 1.07 mg; P=0.027), and GGCX 3261G>A explains 2.3% of the univariate warfarin dose variance. CONCLUSION: GGCX 3261G>A may affect warfarin dose requirements, and showed a small but significant effect on warfarin dose in a Chinese patient population.

[Clinical applications of dosing algorithm in the predication of warfarin maintenance dose].

OBJECTIVE: To evaluate the feasibility of clinical application for genetic based dosing algorithm in the predication of warfarin maintenance dose in Chinese population. METHODS: The clinical data were collected and blood samples harvested from a total of 126 patients undergoing heart valve replacement. The genotypes of VKORC1 and CYP2C9 were determined by melting curve analysis after PCR. They were divided randomly into the study and control groups. In the study group, the first three doses of warfarin were prescribed according to the predicted warfarin maintenance dose while warfarin was initiated at 2.5 mg/d in the control group. The warfarin doses were adjusted according to the measured international normalized ratio (INR) values. And all subjects were followed for 50 days after an initiation of warfarin therapy. RESULTS: At the end of a 50-day follow-up period, the proportions of the patients on a stable dose were 82.4% (42/51) and 62.5% (30/48) for the study and control groups respectively. The mean durations of reaching a stable dose of warfarin were (27.5 ± 1.8) and (34.7 ± 1.8) days and the median durations were (24.0 ± 1.7) and (33.0 ± 4.5) days in the study and control groups respectively. Significant differences existed in the durations of reaching a stable dose between the two groups (P = 0.012). Compared with the control group, the hazard ratio (HR) for the duration of reaching a stable dose was 1.786 in the study group (95%CI 1.088 - 2.875, P = 0.026). CONCLUSION: The predicted dosing algorithm incorporating genetic and non-genetic factors may shorten the duration of achieving efficiently a stable dose of warfarin. And the present study validates the feasibility of its clinical application.

Validation of VKORC1 and CYP2C9 genotypes on interindividual warfarin maintenance dose: a prospective study in Chinese patients.

OBJECTIVES: To develop a warfarin-dosing algorithm that could be combined with pharmacogenomic and demographic factors, and to evaluate its effectiveness in a randomized prospective controlled clinical trial. METHODS: A pharmacogenetics-based dosing model was derived using retrospective data from 266 Chinese patients and multiple linear regression analysis. To prospectively validate this model, 156 patients with an operation of heart valve replacement were enrolled and randomly assigned to the group of pharmacogenetics-guided or traditional dosing for warfarin therapy. All patients were followed up for 50 days after initiation of warfarin therapy. The log-rank test was compared with the time-to-event (Kaplan-Meier) curves. Cox proportional hazards-regression model was used to assess the hazard ratio of the time to reach stable dose. RESULTS: The linear regression model derived from the pharmacogenomic model correlated with 54.1% of warfarin dosing variance. The final multiple linear regression model included age, body surface area, VKORC1, and CYP2C9 genotype. The study showed that the hazard ratio for the time to reach stable dose was 1.932 for the traditional dosing group versus the model-based group and a close and highly significant relationship was observed to exist between the predicted and the actual warfarin dose (R=0.454). CONCLUSION: A pharmacogenetics-based dosing algorithm has been developed for improvement in the time to reach the stable dosing of warfarin. This model may be useful in helping the clinicians to prescribe warfarin with greater safety and efficiency.

[Myocardial protective effect of L-carnitine in cardioplegia solution on patients undergoing heart valve replacement operation].

OBJECTIVE: To investigate the myocardial protective effect of L-carnitine as an ingredient of cardiac arresting solution in the process of heart valve replacement operation. METHODS: 69 cases undergoing heart valve replacement with cardiopulmonary bypass (CPB), 47 males and 22 females, aged 48.17 +/- 14.22 (16 approximately 74 years), were divided into 3 groups: test group I (n = 22, 12 g/L L-carnitine was put in the St. Thomas II cold crystal cardiac arresting liquid), test group II (n = 24, 6 g/L L-carnitine was put in the St. Thomas II cold crystal cardiac arresting liquid), and control group (n = 23, no L-carnitine was put in the St. Thomas II cold crystal cardiac arresting liquid). Before operation, 20 minutes after the beginning of shunt, after the finish of shunt, and 8 hours, one day, 3 days, and 7 days after operation venous blood was drawn to test the serum cardial tropnin I (cTnI), aspartate transaminase, lactate dehydrogenase, creatine kinase (CK) and CK-MB isozyme. Heart color ultrasonography was conducted to test the cardiac index (CI) and left heart ejecting fraction (EF) one day before operation and 7 days after operation. Before shunt and by the end of intracardiac procedure a bit of myocardial tissue was taken to undergo electron microscopy. The amounts of vaso-active drugs, such as dopamine and dobutamine, used postoperatively, and the postoperative cardiac auto-rebeating rate were recorded. RESULTS: The Amounts of vaso-active drugs used after operation was 329 +/- 54 mg in the test group I and 339 +/- 47 mg in the test group II, both significantly less than in the control group (669 +/- 56 mg, both P < 0.01) without a significant difference between the 2 test groups. Since the end of CPB to 3 days after operation, the serum levels of cTnI, aspartate transaminase, lactate dehydrogenase, CK and CK-MB isozyme were significantly lower in the 2 test groups than in the control group (P < 0.05 or P < 0.01). The serum level of cTnI in test group I was significantly lower than in the test group II (5.71 +/- 1.14 ng/ml vs 7.87 +/- 1.89 ng/ml 1 day postoperatively (P < 0.05), and 5.01 +/- 0.89 ng/ml vs 7.53 +/- 1.43 ng/ml 3 days postoperatively (P < 0.05). The postoperative cardiac auto-rebeating rate was 87.9% in the test group I and 74.3% in the test group II, both significantly higher than that in the control group (45.7%, P < 0.05 and P < 0.01). Heart color ultrasonogram showed that 7 days postoperatively the CI index was 2.86 +/- 0.55 and 2.74 +/- 0.56 in the 2 test groups, significantly higher than that in the control group (2.11 +/- 0.35, both P < 0.05), and the left heart EF were 64.3 +/- 8.6 and 59.1 +/- 6.7 in the 2 test groups, both significantly higher than that in the control group (51.7 +/- 4.9, both P < 0.05). Electron microscopy showed only slight swelling of mitochondria in the cardial cell and the myocardial fiber was intact by the end of operation in the 2 test groups without significant difference between these 2 groups, however, in the control group swelling of mitochondria with vesicle formation, fissure of part of mitochondrial ridges, and disappearance of glycogen particles were found. CONCLUSION: Antegrade coronary perfusion of L-carnitine has a good protective effect on myocardium and is worth spreading for heart valve replacement patients with cardiopulmonary bypass.