Functional Analysis of an Intronic FBN1 Pathogenic Gene Variant in a Family With Marfan Syndrome.

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder that canonically affects the ocular, skeletal, and cardiovascular system, in which aortic tear and rupture is the leading cause of death for MFS patients. Genetically, MFS is primarily associated with fibrillin-1 (FBN1) pathogenic variants. However, the disease-causing variant in approximately 10% of patients cannot be identified, partly due to some cryptic mutations that may be missed using routine exonic sequencing, such as non-coding intronic variants that affects the RNA splicing process. We present a 32-year female with typical MFS systemic presentation that reached to a clinical diagnosis according to the revised Ghent nosology. We performed whole-exome sequencing (WES) but the report failed to identify known causal variants when analyzing the exonic sequence. However, further investigation on the exon/intron boundaries of the WES report revealed a candidate intronic variant of the fibrillin 1 (FBN1) gene (c.248-3 C>G) that predicted to affect the RNA splicing process. We conducted minigene splicing analyses and demonstrated that the c.248-3 C>G variant abolished the canonical splicing site of intron 3, leading to activation of two cryptic splicing sites and causing insertion (c.248-1_248-2insAG and c.248-1_248-282ins). Our study not only characterizes an intronic variant to the mutational spectrum of the FBN1 gene in MFS and its aberrant effect on splicing, but highlights the importance to not neglect the exon/intron boundaries when reporting and assessing WES results. We point out the need of conducting functional analysis to verify the pathogenicity of intronic mutation, and the opportunity to re-consider the standard diagnostic approaches in cases of clinically diagnosed MFS with normal or variant of unknown significance genetic results.

microRNA-363-3p reduces endothelial cell inflammatory responses in coronary heart disease via inactivation of the NOX4-dependent p38 MAPK axis.

Coronary heart disease (CHD) is one of the leading causes of heart-associated deaths worldwide. This study aimed to investigate the mechanism by which microRNA-363-3p (miR-363-3p) regulates endothelial injury induced by inflammatory responses in CHD. The expression patterns of miR-363-3p, NADPH oxidase 4 (NOX4), and p38 MAPK/p-p38 MAPK were examined in an established atherosclerosis (AS) model in C57BL/6 mice and in isolated coronary arterial endothelial cells (CAECs) after gain- or loss-of-function experiments. We also measured the levels of inflammatory factors (IL-6, ICAM-1, IL-10 and IL-1ß), hydrogen peroxide (H2O2), and catalase (CAT) activity, followed by detection of cell viability and apoptosis. In AS, miR-363-3p was downregulated and NOX4 was upregulated, while miR-363-3p was identified as targeting NOX4 and negatively regulating its expression. The AS progression was reduced in NOX4 knockout mice. Furthermore, miR-363-3p resulted in a decreased inflammatory response, oxidative stress, and cell apoptosis in CAECs while augmenting their viability via blockade of the p38 MAPK signaling pathway. Overall, miR-363-3p hampers the NOX4-dependent p38 MAPK axis to attenuate apoptosis, oxidative stress injury, and the inflammatory reaction in CAECs, thus protecting CAECs against CHD. This finding suggests the miR-363-3p-dependent NOX4 p38 MAPK axis as a promising therapeutic target for CHD.

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.

Evaluation of the algorithm of Anticlot Assistant: an anticoagulant management system based on mobile health technology.

The purpose is to evaluate the algorithm of Anticlot Assistant, a novel anticoagulant management system based on mobile health technology which was developed to facilitate patient self-management. The eligible patients managed warfarin therapy with usual care, following the prescriptions of the doctors. The actual prescriptions of doctors and the virtual recommendations by Anticlot Assistant were compared and analyzed. There were no significant differences between the next test dates recommended by Anticlot Assistant and those prescribed by doctors. The mean warfarin dosage prescribed by doctors was lower than that recommended by Anticlot Assistant (2.74 ±â€Š1.17 vs. 2.79 ±â€Š1.21 mg, 95% confidence interval for the difference: -0.01--0.09, P = 0.019, n = 139), resulting in the international normalized ratio a high time below the therapeutic range (TTR) (29.9 ±â€Š17.9%), and a low time above TTR [0.0% (0.0-18.7%)]. A mixed linear model revealed that 'the variations of the dosages prescribed by doctors from those recommended by Anticlot Assistant' were positively correlated with 'variations of next international normalized ratios from TTR' after controlling for other factors (estimate of the effect = 0.231, 95% confidence interval: 0.034-0.428, P = 0.022). Anticlot Assistant can mimic the doctors' prescriptions for the next test date and the warfarin dosages recommended by Anticlot Assistant might be more reasonable than those prescribed by doctors, which indicated that the algorithm was reliable and it was possible for the patients to manage warfarin therapy themselves with the aid of Anticlot Assistant.

Resection of a Giant Bronchogenic Cyst in the Left Atrium.

A bronchogenic cyst in the left atrium is rare. We report the case of a 17-year-old male patient who was admitted to the emergency department because of severe chest pain and dyspnea. He was diagnosed using echocardiography and computed tomography, which revealed a huge cardiac tumour in the dome of the left atrium. He was surgically treated with tumour enucleation. The resultant atrial dome defect was reconstructed with a bovine pericardial patch. Pathologic investigation revealed that the tumour was a bronchogenic benign cyst.

Effects of Isolated Impaired Fasting Glucose on Brain Injury During Cardiac Surgery Under Cardiopulmonary Bypass.

Objective: To evaluate the effects of isolated impaired fasting glucose (IIFG) on brain injury in patients undergoing cardiopulmonary bypass (CPB) surgery. Methods: Patients with rheumatic heart valve disease who underwent elective mitral valve replacement were included and divided into control and IIFG groups. Pre-, intra-, and postoperative blood glucose levels, serum insulin levels, insulin resistance index (HOMA-IR), lactic acid levels, and neuron-specific enolase (NSE) and S100B levels were measured. The cerebral oxygen extraction ratio (OER) was calculated. Cognitive function was assessed via the Mini-Mental State Examination (MMSE). Results: HOMA-IR levels were higher in the IIFG group than the control group 30 min after the beginning of CPB, at the termination of CPB, and 2 h after the termination of CPB. Cerebral OER and lactic acid increased intraoperatively in both groups, especially in the IIFG group. NSE and S100B levels were higher in the IIFG group than in the control group at the termination of CPB, 2 h after the termination of CPB, and at 24 h postoperatively. The MMSE scores did not significantly differ between the two groups. Delirium occurred in two patients in the IIFG group, and one in the control group. No other signs and symptoms of brain injuries were detected in either group. Conclusions: The increased postoperative NSE and S100B levels in the IIFG group compared with controls may be associated with severe insulin resistance and stress hyperglycemia. However, the IIFG group did not have clinical manifestations of brain injuries, including cognitive impairment.

MicroRNA-204 as an Indicator of Severity of Pulmonary Hypertension in Children with Congenital Heart Disease Complicated with Pulmonary Hypertension.

BACKGROUND The objective of this study was to investigate the changes and significance of microRNA-204 (miR-204) in children with congenital heart disease (CHD) complicated with pulmonary hypertension (PH). MATERIAL AND METHODS Fifty-two CHD patients with left-to-right shunt were divided into 3 groups according to preoperative pulmonary artery systolic pressure (PASP) detected by color Doppler echocardiography: a control group (PASP <30 mmHg), a mild PH group (PASP 30-49 mmHg), and a severe PH group (PASP >50 mmHg). Peripheral venous blood and supernatant were collected on an empty stomach at 1 h before surgery and 7 days after surgery. The expression of miR-204 in plasma was detected by RT-qPCR. RESULTS One hour before surgery and 7 days after surgery, plasma miR-204 expression was at a higher level than that in the mild PH group and higher than in the severe PH group. miR-204 expression in children in each group showed a decreasing trend after surgery. The mild PH and severe PH groups had lower plasma miR-204 expression and PASP after surgery than before surgery. In the mild PH and severe PH groups, plasma miR-204 expression was negatively correlated with PASP. In all 52 cases, plasma miR-204 expression was negatively correlated with PASP. CONCLUSIONS The plasma miR-204 expression in CHD children with PH was negatively correlated with the degree of PH, suggesting miR-204 may be involved in PH development, and miR-204 expression may be an indicator of PH severity.

Mitigating effect of tanshinone IIA on ventricular remodeling in rats with pressure overload-induced heart failure.

PURPOSE: To investigate the effect of tanshinone IIA (TIIA) on ventricular remodeling in rats with pressure overload-induced heart failure. METHODS: Pressure overload-induced heart failure model (abdominal aortic coarctation) was established in 40 rats, which were divided into model and 5, 10 and 20 mg/kg TIIA groups. Ten rats receiving laparotomy excepting abdominal aortic coarctation were enrolled in sham-operated group. The 5, 10 and 20 mg/kg TIIA groups were treated with 5, 10 and 20 mg/kg TIIA, respectively, for 8 weeks. RESULTS: Compared with model group, in 20 mg/kg TIIA group the left ventricular ejection fraction, left ventricular fractional shortening, left ventricular systolic pressure, ±maximum left ventricular pressure rising and dropping rate, and myocardial B-cell lymphoma-2 and cleaved cysteinyl aspartate specific proteinase-3 protein levels were increased, respectively (P<0.05), and the left ventricular end diastolic diameter, left ventricular end systolic diameter, left ventricular end diastolic pressure, heart weight index, left ventricular weight index, serum B-type brain natriuretic peptide, interleukin 6 and C-reactive protein levels and myocardial B-cell lymphoma-2 associated X protein level were decreased, respectively (P<0.05). CONCLUSION: TIIA may alleviate ventricular remodeling in rats with pressure overload-induced heart failure heart by reducing inflammatory response and cardiomyocyte apoptosis.

Patient compliance with an anticoagulation management system based on a smartphone application.

We developed a novel anticoagulation management system (Anticlot Assistant) based on a smartphone application (App). This study was performed to evaluate patient compliance with Anticlot Assistant. This prospective case series study involved patients receiving warfarin therapy. The eligible patients were managed via Anticlot Assistant, and outcome data were analyzed. Thirty patients were recruited. The mean time within the therapeutic range (TTR) was 56.5% ± 26.2% and the mean patient compliance with Anticlot Assistant was 52.7% ± 40.4%. The patients in good compliance group had higher TTR (65.6 ± 25.0% vs. 40.0 ± 21.0%, P = 0.009), lower time in the extremely low range (9.4 ± 10.6% vs. 27.4 ± 13.2%, P = 0.000) and in the extremely high range (1.3 ± 2.8% vs. 14.1 ± 22.3%, P = 0.004) than those in poor compliance group. Logistic regression analysis revealed that receiving an education of > 6 years was the only independent predictor of good compliance (odds ratio 8.400, 95% confidence interval 1.274-55.394, P = 0.027). Patient compliance is critical important for good outcomes and it might increase with improvements in education and more widespread use of information technology. Although further improvement is needed, Anticlot Assistant is promising and this study offered valuable experiences for further research.

LncRNA XIST regulates myocardial infarction by targeting miR-130a-3p.

The study was used to probe long noncoding RNA X-inactive specific transcript (lncRNA XIST) RNA expression profile and its influence on cell cycle, proliferation, and apoptosis in myocardial cells. We also aimed to explore the possible meditating relationship between XIST, PDE4D, and miR-130a-3p. Gene differential analysis was carried out using human lncRNA Microarray V3.0. quantitative real-time PCR was used to test mRNA expressions of XIST, miR-130a-3p, and PDE4D in normal cells and postmyocardial infarction (MI) cells. Western blot was applied to determine the protein expression profile of PED4D. Changes in viability and cell cycle/apoptosis of post-MI myocardial cells after silencing of XIST or PDE4D were investigated by MTT assay and flow cytometry, respectively. The targeting relationship between miR-130a-3p and XIST, PDE4D in myocardial cells were verified by dual luciferase reporter assay. Simulated MI environment was constructed by performing anoxic preconditioning in normal cells to probe the influence of XIST on myocardial cell apoptosis. XIST and PDE4D were overexpressed in post-MI myocardial cells, whereas miR-130a-3p was underexpressed in post-MI myocardial cells. High-expressed XIST and PDE4D both promoted myocardial cell apoptosis. High-expressed XIST also inhibited myocardial cell proliferation. XIST-downregulated miR-130a-3p and PDE4D was a direct target of miR-130a-3p. LncRNA XIST promotes MI by targeting miR-130a-3p. MI induced by PDE4D can be reversed by miR-130a-3p.

Mitigating effect of tanshinone IIA on ventricular remodeling in rats with pressure overload-induced heart failure

Abstract Purpose To investigate the effect of tanshinone IIA (TIIA) on ventricular remodeling in rats with pressure overload-induced heart failure. Methods Pressure overload-induced heart failure model (abdominal aortic coarctation) was established in 40 rats, which were divided into model and 5, 10 and 20 mg/kg TIIA groups. Ten rats receiving laparotomy excepting abdominal aortic coarctation were enrolled in sham-operated group. The 5, 10 and 20 mg/kg TIIA groups were treated with 5, 10 and 20 mg/kg TIIA, respectively, for 8 weeks. Results Compared with model group, in 20 mg/kg TIIA group the left ventricular ejection fraction, left ventricular fractional shortening, left ventricular systolic pressure, ±maximum left ventricular pressure rising and dropping rate, and myocardial B-cell lymphoma-2 and cleaved cysteinyl aspartate specific proteinase-3 protein levels were increased, respectively (P<0.05), and the left ventricular end diastolic diameter, left ventricular end systolic diameter, left ventricular end diastolic pressure, heart weight index, left ventricular weight index, serum B-type brain natriuretic peptide, interleukin 6 and C-reactive protein levels and myocardial B-cell lymphoma-2 associated X protein level were decreased, respectively (P<0.05). Conclusion TIIA may alleviate ventricular remodeling in rats with pressure overload-induced heart failure heart by reducing inflammatory response and cardiomyocyte apoptosis.

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.

The protective effect of umbelliferone ameliorates myocardial injury following ischemia­reperfusion in the rat through suppression NLRP3 inflammasome and upregulating the PPAR-γ.

The present study investigated whether the protective effect of umbelliferone could regulate myocardial injury following ischemia­reperfusion and improve mitochondrial respiratory function, thereby relieving myocardial injury following ischemia­reperfusion in rats. In the present study, the extent of inflammation and oxidative stress were analyzed using ELISA. Western blot analysis was employed to investigate the protein expression levels of the PYD domains­containing protein 3 (NLRP3) inflammasome and peroxisome proliferator­activated receptor-γ (PPAR­Î³). Compared with the myocardial injury following ischemia­reperfusion group, umbelliferone significantly prevented myocardial injury, inhibited oxidative stress markers (superoxide dismutase and malondialdehyde), reduced inflammation (tumor necrosis factor­α and interleukin­6) and myocardial apoptosis levels (caspase­3/9 and apoptosis regular B­cell lymphoma­2­associated X protein) in the myocardial injury following ischemia­reperfusion group of rats. Umbelliferone treatment also suppressed NACHT, LRR and NLRP3 inflammasome activation and induced PPAR­Î³ expression. The results of the present study suggested that the protective effect of umbelliferone may ameliorate myocardial injury following ischemia­reperfusion in the rat through the suppression of the NLRP3 inflammasome and upregulating PPAR­Î³ expression.

Delayed protective effect of telmisartan on lung ischemia/reperfusion injury in valve replacement operations.

The present study aimed to investigate the delayed protective effect of telmisartan on lung ischemic/reperfusion injury in patients undergoing heart valve replacement operations. In total, 180 patients diagnosed with rheumatic valve diseases were randomly divided into the telmisartan (T), captopril (C) and placebo (P) groups. In the telmisartan group, the patients were pretreated with telmisartan (1 mg/kg/day), at the time period 96-48 h before the operation, whereas in the C group, the patients were treated with captopril (1 mg/kg/day) at the time period 96-48 h prior to the operation control group. Each drug treatment group included a corresponding placebo treatment. The variables pulmonary vascular resistance (PVR) and A-aDO2 were measured prior to CPB and at 1, 3, 6 and 12 h after CPB. Pulmonary neutrophil (PMN) count in the left and right atrium blood as well as SOD malondialdehyde (MDA), NO, angiotensin II (AngII) value in the left atrium blood, were measured 30 min prior to and after CPB. The PVR parameters of the telmisartan and captopril groups were significantly lower than those of the placebo group (P<0.05). The A-aDO2 values in the telmisartan and captopril groups were significantly lower than those in the placebo group at 1, 3 and 6 h following CPB treatment. The difference between the right and left atrium blood PMN was significantly lower in the telmisartan and captopril intervention groups compared to that in the placebo group 30 min following CPB treatment. The left atrium blood SOD and NO values were significantly higher, whereas the MDA value was significantly lower in the telmisartan group compared to the control group 30 min following CPB treatment. As for AngII, there was no difference between the C and T groups, compared with the P group. In the two groups 30 min after treatment with CPB, 24 patients experienced varying degrees of cough, with the telmisartan group showing a significant difference (P<0.05). The hospitalization time was compared in the three groups of patients and it was found to be significantly shorter in the telmisartan group than the captopril and placebo groups (P<0.05). In conclusion, it was found that for the time period 96-48 h before heart valve replacement operations telmisartan (1 mg/kg/day) delayed the protective effect on lung ischemia/reperfusion injury in patients with rheumatic valve diseases. The results of the present study indicated that the protective effect may be associated with the increment of endogenetic NO and the enhanced ability against lipid peroxidation.

Effects of Tissue-Type Plasminogen Site-Specific Transgene in Gelatin-Coated Dacron on the Fibrinolysis Activity of Rabbit Left Atrium.

BACKGROUND: To investigate the effects of tissue-type plasminogen activator (tPA) gene transfer with left-atrium local positioning on the fibrinolytic activity of rabbit left atrial blood. METHODS: A total of 48 rabbits were randomly divided into 3 groups (n = 16): gene therapy, vector control, and blank control groups. Each group was divided into 2 subgroups (8 rabbits in each subgroup) according to the sacrifice time on the postoperative 3(rd) and 14(th) days. The tPA mRNA transcriptional level and exogenous tPA protein expression within regional myocardial tissues of the left atrium were detected on the postoperative 3(rd) and 14(th) days. After excluding the animals that died, 6 samples of each subgroup were randomly selected for the statistics (n = 6). RESULTS: The tPA activities in rabbit left atrial blood and peripheral blood were also detected. The tPAmRNA and tPA protein expressions within regional myocardial tissues were detected on the postoperative 3(rd) and 14(th) days. The tPA activity in left atrial blood in the gene therapy group was higher than the tPA activity of other groups (p < 0.02). No significant differences were observed in the tPA activity of peripheral blood among the 3 groups before surgery. A gelatin-coated Dacron piece, which carried the tPA gene, was implanted in the left atrial appendage. CONCLUSIONS: The gelatin-coated Dacron piece could express and secrete tPA proteins in the region, thus enhancing the fibrinolytic activity of left atrial blood. KEY WORDS: Fibrinolytic activity; Gelatin coating; Gene; Left atrium; Tissue-type plasminogen activator.

[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.