Successful ablation of a right concealed epicardial accessory pathway using ethanol infusion.

INTRODUCTION: This study describes a rare case of concealed epicardial accessory pathway (AP) successfully ablated using ethanol infusion (EI) through a variant vessel connecting the right atrium (RA) and the right ventricle (RV) surface. METHODS AND RESULTS: A 58-year-old male referred to our hospital for prior failed AP ablation. Cardiac-enhanced computerized tomography scan showed there was a variant vessel at the tip of right atrial appendage and a pulmonary artery (PA)-RA fistula at the roof of RA. The earliest activation was present at the site of the PA-RA fistula. A selective angiography showed that a small branch of the variant vessel covered the earliest excitation site of the AP. EI into this branch successfully repressed the AP without any recurrences within a follow-up period of 3 months. CONCLUSION: Endocardial ablation is challenging for epicardial APs related to cardiac structural variations. If small vascular branches near the earliest activation site can be found, EI can successfully ablate these types of epicardial APs.

Novel combinations of variations in KCNQ1 were associated with patients with long QT syndrome or Jervell and Lange-Nielsen syndrome.

OBJECTIVES: Long QT syndrome (LQTS) is one of the primary causes of sudden cardiac death (SCD) in youth. Studies have identified mutations in ion channel genes as key players in the pathogenesis of LQTS. However, the specific etiology in individual families remains unknown. METHODS: Three unrelated Chinese pedigrees diagnosed with LQTS or Jervell and Lange-Nielsen syndrome (JLNS) were recruited clinically. Whole exome sequencing (WES) was performed and further validated by multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing. RESULTS: All of the probands in our study experienced syncope episodes and featured typically prolonged QTc-intervals. Two probands also presented with congenital hearing loss and iron-deficiency anemia and thus were diagnosed with JLNS. A total of five different variants in KCNQ1, encoding a subunit of the voltage-gated potassium channel, were identified in 3 probands. The heterozygous variants, KCNQ1 c.749T > C was responsible for LQTS in Case 1, transmitting in an autosomal dominant pattern. Two patterns of compound heterozygous variants were responsible for JLNS, including a large deletion causing loss of the exon 16 and missense variant c.1663 C > T in Case 2, and splicing variant c.605-2 A > G and frame-shift variant c.1265del in Case 3. To our knowledge, the compound heterozygous mutations containing a large deletion and missense variant were first reported in patients with JLNS. CONCLUSION: Our study expanded the LQTS genetic spectrum, thus favoring disease screening and diagnosis, personalized treatment, and genetic consultation.

Endothelial cell-derived tetrahydrobiopterin prevents aortic valve calcification.

AIMS: Tetrahydrobiopterin (BH4) is a critical determinant of the biological function of endothelial nitric oxide synthase. The present study was to investigate the role of valvular endothelial cell (VEC)-derived BH4 in aortic valve calcification. METHODS AND RESULTS: Plasma and aortic valve BH4 concentrations and the BH4:BH2 ratio were significantly lower in calcific aortic valve disease patients than in controls. There was a significant decrease of the two key enzymes of BH4 biosynthesis, guanosine 5'-triphosphate cyclohydrolase I (GCH1) and dihydrofolate reductase (DHFR), in calcified aortic valves compared with the normal ones. Endothelial cell-specific deficiency of Gch1 in Apoe-/- (Apoe-/-Gch1fl/flTie2Cre) mice showed a marked increase in transvalvular peak jet velocity, calcium deposition, runt-related transcription factor 2 (Runx2), dihydroethidium (DHE), and 3-nitrotyrosine (3-NT) levels in aortic valve leaflets compared with Apoe-/-Gch1fl/fl mice after a 24-week western diet (WD) challenge. Oxidized LDL (ox-LDL) induced osteoblastic differentiation of valvular interstitial cells (VICs) co-cultured with either si-GCH1- or si-DHFR-transfected VECs, while the effects could be abolished by BH4 supplementation. Deficiency of BH4 in VECs caused peroxynitrite formation increase and 3-NT protein increase under ox-LDL stimulation in VICs. SIN-1, the peroxynitrite generator, significantly up-regulated alkaline phosphatase (ALP) and Runx2 expression in VICs via tyrosine nitration of dynamin-related protein 1 (DRP1) at Y628. Finally, folic acid (FA) significantly attenuated aortic valve calcification in WD-fed Apoe-/- mice through increasing DHFR and salvaging BH4 biosynthesis. CONCLUSION: The reduction in endothelial-dependent BH4 levels promoted peroxynitrite formation, which subsequently resulted in DRP1 tyrosine nitration and osteoblastic differentiation of VICs, thereby leading to aortic valve calcification. Supplementation of FA in diet attenuated hypercholesterolaemia-induced aortic valve calcification by salvaging BH4 bioavailability.

Cathepsin B aggravates coxsackievirus B3-induced myocarditis through activating the inflammasome and promoting pyroptosis.

Cathepsin B (CatB) is a cysteine proteolytic enzyme widely expressed in various cells and mainly located in the lysosomes. It contributes to the pathogenesis and development of many diseases. However, the role of CatB in viral myocarditis (VMC) has never been elucidated. Here we generated the VMC model by intraperitoneal injection of coxsackievirus B3 (CVB3) into mice. At day 7 and day 28, we found CatB was significantly activated in hearts from VMC mice. Compared with the wild-type mice receiving equal amount of CVB3, genetic ablation of CatB (Ctsb-/-) significantly improved survival, reduced inflammatory cell infiltration, decreased serum level of cardiac troponin I, and ameliorated cardiac dysfunction, without altering virus titers in hearts. Conversely, genetic deletion of cystatin C (Cstc-/-), which markedly enhanced CatB levels in hearts, distinctly increased the severity of VMC. Furthermore, compared with the control, we found the inflammasome was activated in the hearts of wild-type mice with VMC, which was attenuated in the hearts of Ctsb-/- mice but was further enhanced in Cstc-/- mice. Consistently, the inflammasome-initiated pyroptosis was reduced in Ctsb-/- mice hearts and further increased in Cstc-/- mice. These results suggest that CatB aggravates CVB3-induced VMC probably through activating the inflammasome and promoting pyroptosis. This finding might provide a novel strategy for VMC treatment.

Heme Oxygenase-1 in Macrophages Drives Septic Cardiac Dysfunction via Suppressing Lysosomal Degradation of Inducible Nitric Oxide Synthase.

RATIONALE: To date, our understanding of the role of HO-1 (heme oxygenase-1) in inflammatory diseases has mostly been limited to its catalytic function and the potential for its heme-related catabolic products to suppress inflammation and oxidative stress. Whether and how HO-1 in macrophages plays a role in the development of septic cardiac dysfunction has never been explored. OBJECTIVE: Here, we investigated the role of macrophage-derived HO-1 in septic cardiac dysfunction. METHODS AND RESULTS: Intraperitoneal injection of lipopolysaccharide significantly activated HO-1 expression in cardiac infiltrated macrophages. Surprisingly, we found that myeloid conditional HO-1 deletion in mice evoked resistance to lipopolysaccharide-triggered septic cardiac dysfunction and lethality in vivo, which was accompanied by reduced cardiomyocyte apoptosis in the septic hearts and decreased peroxynitrite production and iNOS (inducible NO synthase) in the cardiac infiltrated macrophages, whereas proinflammatory cytokine production and macrophage infiltration were unaltered. We further demonstrated that HO-1 suppression abolished the lipopolysaccharide-induced iNOS protein rather than mRNA expression in macrophages. Moreover, we confirmed that the inhibition of HO-1 promoted iNOS degradation through a lysosomal rather than proteasomal pathway in macrophages. Suppression of the lysosomal degradation of iNOS by bafilomycin A1 drove septic cardiac dysfunction in myeloid HO-1-deficient mice. Mechanistically, we demonstrated that HO-1 interacted with iNOS at the flavin mononucleotide domain, which further prevented iNOS conjugation with LC3 (light chain 3) and subsequent lysosomal degradation in macrophages. These effects were independent of HO-1's catabolic products: ferrous ion, carbon monoxide, and bilirubin. CONCLUSIONS: Our results indicate that HO-1 in macrophages drives septic cardiac dysfunction. The mechanistic insights provide potential therapeutic targets to treat septic cardiac dysfunction.

Next-generation sequencing identified novel Desmoplakin frame-shift variant in patients with Arrhythmogenic cardiomyopathy.

BACKGROUND: Arrhythmogenic cardiomyopathy (AC) is one of the leading causes for sudden cardiac death (SCD). Recent studies have identified mutations in cardiac desmosomes as key players in the pathogenesis of AC. However, the specific etiology in individual families remains largely unknown. METHODS: A 4-generation family presenting with syncope, lethal ventricular arrhythmia and SCD was recruited. Targeted next generation sequencing (NGS) was performed and validated by Sanger sequencing. Plasmids containing the mutation and wild type (WT) were constructed. Real-time PCR, western-blot and immunofluorescence were performed to detect the functional change due to the mutation. RESULTS: The proband, a 56-year-old female, presented with recurrent palpitations and syncope. An ICD was implanted due to her family history of SCD/ aborted SCD. NGS revealed a novel heterozygous frame-shift variant (c.832delG) in Desmoplakin (DSP) among 5 family members. The variant led to frame-shift and premature termination, producing a truncated protein. Cardiac magnetic resonance (CMR) of the family members carrying the same variant shown myocardium thinning and fatty infiltration in the right ventricular, positive bi-ventricular late gadolinium enhancement and severe RV dysfunction, fulfilling the diagnostic criteria of AC. HEK293T cells transfected with mutant plasmids expressed truncated DSP mRNA and protein, upregulation of nuclear junction plakoglobin (JUP) and downregulation of ß-catenin, when compared with WT. CONCLUSION: We infer that the novel c.832delG variant in DSP was associated with AC in this family, likely through Wnt/ß-catenin signaling pathway.

Does Quicker Mean Better? Comparison of Rapid Deployment Versus Conventional Aortic Valve Replacement.

The aim of this meta-analysis was to compare the clinical outcomes in patients who underwent rapid deployment aortic valve replacement (RDAVR) and conventional bio prosthetic aortic valve replacement (CAVR).We performed a literature search by August 2018. The primary outcomes were hospital and 1-year mortality, and the secondary endpoints included the aortic cross-clamp (ACC), cardiopulmonary bypass (CPB) time, and postoperative and valve-related complications.Two randomized controlled trials and 13 propensity score-matched studies were included. There was no difference between RDAVR and CAVR in hospital mortality (2.5% versus 2.1%; risk ratio (RR) 1.16 [95% confidence interval (CI) 0.80-1.68]) or 1-year mortality (2.9% versus 4.1%; RR 0.69 [95% CI 0.34-1.34]). RDAVR significantly reduced the ACC time ( (mean difference (MD) -24.33 [95% CI -28.35 to -20.32]) and CPB time (MD -21.51 [95% CI -22.83 to -20.20]). The pooled analysis showed that RDAVR doubled the occurrence of permanent pacemaker implantation (8.6% versus 4.3%; RR 2.05 [95% CI 1.62-2.60]). Meanwhile, the blood transfusion amount (MD -1.54 [95% CI -2.22 to -0.86]) and postoperative atrial fibrillation (POAF) occurrence (RR 0.83 [95% CI 0.69-0.99]) was reduced. The difference of paravalvular leakage frequency between RDAVR and CAVR was marginal (RR 1.77 [95% CI 1.00-3.17]; P = 0.05). Furthermore, RDAVR was related to larger valves (MD 0.70 cm [95% CI 0.33-1.07]) and lower mean pressure gradients (MD -1.93 mmHg [95% CI -3.58 to -0.28]).The hospital and 1-year survival rates between RDAVR and CAVR are comparable. RDAVR reduces POAF occurrence and blood transfusion but is associated with a higher occurrence of pacemaker implantation.

EphrinB2 Regulates Cardiac Fibrosis Through Modulating the Interaction of Stat3 and TGF-ß/Smad3 Signaling.

RATIONALE: Cardiac fibrosis is a common feature in left ventricular remodeling that leads to heart failure, regardless of the cause. EphrinB2 (erythropoietin-producing hepatoma interactor B2), a pivotal bidirectional signaling molecule ubiquitously expressed in mammals, is crucial in angiogenesis during development and disease progression. Recently, EphrinB2 was reported to protect kidneys from injury-induced fibrogenesis. However, its role in cardiac fibrosis remains to be clarified. OBJECTIVE: We sought to determine the role of EphrinB2 in cardiac fibrosis and the underlying mechanisms during the pathological remodeling process. METHODS AND RESULTS: EphrinB2 was highly expressed in the myocardium of patients with advanced heart failure, as well as in mouse models of myocardial infarction and cardiac hypertrophy induced by angiotensin II infusion, which was accompanied by myofibroblast activation and collagen fiber deposition. In contrast, intramyocardial injection of lentiviruses carrying EphrinB2-shRNA ameliorated cardiac fibrosis and improved cardiac function in mouse model of myocardial infarction. Furthermore, in vitro studies in cultured cardiac fibroblasts demonstrated that EphrinB2 promoted the differentiation of cardiac fibroblasts into myofibroblasts in normoxic and hypoxic conditions. Mechanistically, the profibrotic effect of EphrinB2 on cardiac fibroblast was determined via activating the Stat3 (signal transducer and activator of transcription 3) and TGF-ß (transforming growth factor-ß)/Smad3 (mothers against decapentaplegic homolog 3) signaling. We further determined that EphrinB2 modulated the interaction between Stat3 and Smad3 and identified that the MAD homology 2 domain of Smad3 and the coil-coil domain and DNA-binding domain of Stat3 mediated the interaction. CONCLUSIONS: This study uncovered a previously unrecognized profibrotic role of EphrinB2 in cardiac fibrosis, which is achieved through the interaction of Stat3 with TGF-ß/Smad3 signaling, implying a promising therapeutic target in fibrotic diseases and heart failure.

Increased myocardial stiffness activates cardiac microvascular endothelial cell via VEGF paracrine signaling in cardiac hypertrophy.

When the heart is subjected to an increased workload, mechanical stretch together with neurohumoral stimuli activate the "fetal gene program" and induce cardiac hypertrophy to optimize output. Due to a lack of effective methods/models to quantify and modulate cardiac mechanical properties, the connection between these properties and the development of cardiac hypertrophy remains largely unexplored. Here, we utilized an atomic force microscope (AFM) to directly measure the elastic modulus of the hypertrophic myocardium induced by pressure overload. Additionally, we investigated the effects of extracellular elasticity on angiogenesis, which provides blood and nutrition to support cardiomyocyte hypertrophic growth in this process. In response to pressure overload, the myocardium rapidly developed hypertrophy and correspondingly demonstrated a high elastic modulus property. This mechanical feature correlated with enhanced angiogenesis. Mechanistically, we found that a high elastic modulus promoted cultured cardiomyocytes to synthesize and paracrine vascular endothelial growth factor (VEGF) to activate cardiac microvascular endothelial cells. Further analysis showed that the increased elastic modulus enhanced the interaction between Talin1 and integrin ß1 to activate the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/hypoxia-inducible factor 1α (Hif-1α) pathway, which contributed to VEGF production. Thus, our study revealed a critical role of the elastic modulus in regulating angiogenesis during the development of cardiac hypertrophy.

AMP-activated protein kinase α1 promotes atherogenesis by increasing monocyte-to-macrophage differentiation.

Monocyte-to-macrophage differentiation, which can be initiated by physiological or atherogenic factors, is a pivotal process in atherogenesis, a disorder in which monocytes adhere to endothelial cells and subsequently migrate into the subendothelial spaces, where they differentiate into macrophages and macrophage-derived foam cells and cause atherosclerotic lesions. However, the monocyte-differentiation signaling pathways that are activated by atherogenic factors are poorly defined. Here we report that the AMP-activated protein kinase α1 (AMPKα1) in monocytes promotes atherosclerosis by increasing monocyte differentiation and survival. Exposure of monocytes to oxidized low-density lipoprotein, 7-ketocholesterol, phorbol 12-myristate 13-acetate, or macrophage colony-stimulated factor (M-CSF) significantly activated AMPK and promoted monocyte-to-macrophage differentiation. M-CSF-activated AMPK is via M-CSF receptor-dependent reactive oxygen species production. Consistently, genetic deletion of AMPKα1 or pharmacological inhibition of AMPK blunted monocyte-to-macrophage differentiation and promoted monocyte/macrophage apoptosis. Compared with apolipoprotein E knock-out (ApoE-/-) mice, which show impaired clearing of plasma lipoproteins and spontaneously develop atherosclerosis, ApoE-/-/AMPKα1-/- mice showed reduced sizes of atherosclerotic lesions and lesser numbers of macrophages in the lesions. Furthermore, aortic lesions were decreased in ApoE-/- mice transplanted with ApoE-/-/AMPKα1-/- bone marrow and in myeloid-specific AMPKα1-deficient ApoE-/- mice. Finally, rapamycin treatment, which abolished delayed monocyte differentiation in ApoE-/-/AMPKα1-/- mice, lost its atherosclerosis-lowering effects in these mice. Mechanistically, we found that AMPKα1 regulates FoxO3-dependent expression of both LC3 and ULK1, which are two important autophagy-related markers. Rapamycin treatment increased FoxO3 activity as well as LC3 and ULK1 expressions in macrophages from AMPKα1-/- mice. Our results reveal that AMPKα1 deficiency impairs autophagy-mediated monocyte differentiation and decreases monocyte/macrophage survival, which attenuates atherosclerosis in ApoE-/- mice in vivo.

AMP-Activated Protein Kinase Alpha 2 Deletion Induces VSMC Phenotypic Switching and Reduces Features of Atherosclerotic Plaque Stability.

RATIONALE: AMP-activated protein kinase (AMPK) has been reported to play a protective role in atherosclerosis. However, whether AMPKα2 controls atherosclerotic plaque stability remains unknown. OBJECTIVE: The aim of this study was to evaluate the impact of AMPKα2 deletion on atherosclerotic plaque stability in advanced atherosclerosis at the brachiocephalic arteries and to elucidate the underlying mechanisms. METHODS AND RESULTS: Features of atherosclerotic plaque stability and the markers for contractile or synthetic vascular smooth muscle cell (VSMC) phenotypes were monitored in the brachiocephalic arteries from Apoe(-/-)AMPKα2(-/-) mice or VSMC-specific AMPKα2(-/-) mice in an Apoe(-/-) background (Apoe(-/-)AMPKα2(sm-/-)) fed Western diet for 10 weeks. We identified that Apoe(-/-)AMPKα2(-/-) mice and Apoe(-/-)AMPKα2(sm-/-) mice exhibited similar unstable plaque features, aggravated VSMC phenotypic switching, and significant upregulation of Kruppel-like factor 4 (KLF4) in the plaques located in the brachiocephalic arteries compared with those found in Apoe(-/-) and Apoe(-/-)AMPKα2(sm+/+) control mice. Pravastatin, an AMPK activator, suppressed VSMC phenotypic switching and alleviated features of atherosclerotic plaque instability in Apoe(-/-)AMPKα2(sm+/+) mice, but not in Apoe(-/-)AMPKα2(sm-/-) mice. VSMC isolated from AMPKα2(-/-) mice displayed a significant reduction of contractile proteins(smooth muscle actin-α, calponin, and SM-MHC [smooth muscle-mysion heavy chain]) in parallel with increased detection of synthetic proteins (vimentin and osteopontin) and KLF4, as observed in vivo. KLF4-specific siRNA abolished AMPKα2 deletion-induced VSMC phenotypic switching. Furthermore, pharmacological or genetic inhibition of nuclear factor-κB significantly decreased KLF4 upregulation in VSMC from AMPKα2(-/-) mice. Finally, we found that AMPKα2 deletion markedly promoted the binding of nuclear factor-κBp65 to KLF4 promoter. CONCLUSIONS: This study demonstrated that AMPKα2 deletion induces VSMC phenotypic switching and promotes features of atherosclerotic plaque instability in nuclear factor-κB-KLF4-dependent manner.

Ablation of Adenosine Monophosphate-Activated Protein Kinase α1 in Vascular Smooth Muscle Cells Promotes Diet-Induced Atherosclerotic Calcification In Vivo.

RATIONALE: Atherosclerotic calcification is highly linked with plaque rapture. How calcification is regulated is poorly characterized. OBJECTIVE: We sought to determine the contributions of AMP-activated protein kinase (AMPK) in atherosclerotic calcification. METHODS AND RESULTS: Aortic calcification was evaluated in aortic roots and brachiocephalic arteries of atherosclerotic prone ApoE(-/-) mice or in mice with dual deficiencies of ApoE and AMPKα isoforms in whole body (ApoE(-/-)/AMPKα1(-/-) and ApoE(-/-)/AMPKα2(-/-)) or vascular smooth muscle cell (VSMC)-specific or macrophage-specific knockout of AMPKα1 fed with Western diet for 24 weeks. Genetic deficiency of AMPKα1 but not of AMPKα2 promoted atherosclerotic calcification and the expression of Runx2 (Runt-related transcription factor). Conversely, chronic administration of metformin, which activated AMPK, markedly reduced atherosclerotic calcification and Runx2 expression in ApoE(-/-) mice but had less effects in ApoE(-/-)/AMPKα1(-/-) mice. Furthermore, VSMC-specific but not macrophage-specific ablation of AMPKα1 promoted aortic calcification in vivo. Ablation of AMPKα1 in VSMC prevented Runx2 from proteasome degradation in parallel with aberrant osteoblastic differentiation of VSMC, whereas AMPK activation promoted Runx2 post-translational modification by small ubiquitin-like modifier (SUMO, SUMOylation), which is associated with its instability. Mechanically, we found that AMPKα1 directly phosphorylated protein inhibitor of activated STAT-1 (PIAS1), the SUMO E3-ligase of Runx2, at serine 510, to promote its SUMO E3-ligase activity. Finally, mutation of protein inhibitor of activated STAT-1 at serine 510 suppressed metformin-induced Runx2 SUMOylation and subsequently prevented metformin's effect on reducing oxidized low-density lipoprotein-triggered Runx2 expression in VSMC. CONCLUSIONS: AMPKα1 phosphorylated protein inhibitor of activated STAT-1 to promote Runx2 SUMOylation and subsequently lead to its instability. AMPKα1 deficiency in VSMC increased Runx2 expression and promoted atherosclerotic calcification in vivo.

Interleukin-17A mediates cardiomyocyte apoptosis through Stat3-iNOS pathway.

Interleukin-17A, a pro-inflammatory cytokine, has a direct proapoptotic effect on cardiomyocytes. However, the specific mechanism has not been clarified. In the present study, an in-vitro model of cardiomyocyte apoptosis induced by IL-17A stimulation was employed and the roles of iNOS and Stat3 involved were investigated. Our data showed that the neonatal mouse cardiomyocytes express IL-17 receptors: IL-17RA and IL-17RC, but did not express IL-17A. Exogenous IL-17A significantly induces iNOS expression and hence the cardiomyocyte apoptosis. Moreover, IL-17A-induced cardiomyocyte apoptosis can be achieved directly via iNOS activation. We further showed that exogenous IL-17A simultaneously triggers Stat3 activation, which in turn inhibits IL-17A-induced iNOS expression in cardiomyocytes. And both ChIP and dual-luciferase results confirmed that Stat3 directly inhibits transcriptional activities of iNOS via binding to its specific promoter region. Consistent with these data, silencing of Stat3 in fact can aggravate IL-17A-triggered cardiomyocyte apoptosis. Finally, using an in vivo myocardial ischemia/reperfusion injury model, we verified that Stat3 inhibition increased iNOS expression and exacerbated cardiomyocyte apoptosis. Thus, our data strongly support the notion that Stat3 plays a compensatory anti-apoptotic role in IL-17A/iNOS-mediated cardiomyocyte apoptosis via inhibiting iNOS transcription, providing a novel molecular mechanism of apoptosis regulation and complicated interactions between IL-17A/iNOS and IL-17A/Stat3 signalings.

RAGE deficiency alleviates aortic valve calcification in ApoE-/- mice via the inhibition of endoplasmic reticulum stress.

Receptor for advanced glycation end products (RAGE) and endoplasmic reticulum (ER) stress have been shown to be involved in calcific aortic valve disease (CAVD). However, the association between RAGE and ER stress remains unknown in the pathogenesis of CAVD. The current study aims to test the hypothesis that RAGE deficiency alleviates aortic valve calcification via the inhibition of ER stress. Up-regulation of RAGE and ER stress markers in calcified human aortic valves were confirmed by immunoblotting. Aortic valve calcification was evaluated in atherosclerotic prone ApoE-/- mice or in mice with dual deficiencies of ApoE and RAGE (ApoE-/-RAGE-/-) fed with high cholesterol diet for 24weeks. Echocardiography and histological examination show that genetic deficiency of RAGE attenuates aortic valve calcification in ApoE-/- mice. Meanwhile, RAGE deficiency inhibited the osteogenic signaling and ER stress activation as well as suppressed macrophage infiltration in vivo. Cultured human aortic valve interstitial cells (AVICs) were treated with high molecular group box 1 protein (HMGB1) as in vitro model. We found that HMGB1 induced osteoblastic differentiation and calcification through RAGE/ER stress. Furthermore, Sox9 up-regulation and intranuclear translocation mediated the pro-osteogenic effect of HMGB1 on AVICs. RAGE or ER stress knockdown reduced the up-regulation of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) in human AVICs exposed to HMGB1.These novel findings demonstrate that RAGE deficiency protects against aortic valve calcification in high cholesterol diet-fed ApoE-/- mice via inhibition of ER stress. HMGB1 induces AVIC osteoblastic differentiation and calcification through RAGE/ER stress/Sox9 pathway.

Endothelial Nitric Oxide Synthase-Derived Nitric Oxide Prevents Dihydrofolate Reductase Degradation via Promoting S-Nitrosylation.

OBJECTIVE: Dihydrofolate reductase (DHFR) is a key protein involved in tetrahydrobiopterin (BH4) regeneration from 7,8-dihydrobiopterin (BH2). Dysfunctional DHFR may induce endothelial nitric oxide (NO) synthase (eNOS) uncoupling resulting in enzyme production of superoxide anions instead of NO. The mechanism by which DHFR is regulated is unknown. Here, we investigate whether eNOS-derived NO maintains DHFR stability. APPROACH AND RESULTS: DHFR activity, BH4 content, eNOS activity, and S-nitrosylation were assessed in human umbilical vein endothelial cells and in aortas isolated from wild-type and eNOS knockout mice. In human umbilical vein endothelial cells, depletion of intracellular NO by transfection with eNOS-specific siRNA or by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)-both of which had no effect on DHFR mRNA levels-markedly reduced DHFR protein levels in parallel with increased DHFR polyubiquitination. Supplementation of S-nitroso-l-glutathione (GSNO), a NO donor, or MG132, a potent inhibitor of the 26S proteasome, prevented eNOS silencing and PTIO-induced DHFR reduction in human umbilical vein endothelial cells. PTIO suppressed S-nitrosylation of DHFR, whereas GSNO promoted DHFR S-nitrosylation. Mutational analysis confirmed that cysteine 7 of DHFR was S-nitrosylated. Cysteine 7 S-nitrosylation stabilized DHFR from ubiquitination and degradation. Experiments performed in aortas confirmed that PTIO or eNOS deficiency reduces endothelial DHFR, which can be abolished by MG132 supplementation. CONCLUSIONS: We conclude that S-nitrosylation of DHFR at cysteine 7 by eNOS-derived NO is crucial for DHFR stability. We also conclude that NO-induced stabilization of DHFR prevents eNOS uncoupling via regeneration of BH4, an essential eNOS cofactor.

Oxidized low-density lipoprotein promotes osteoblastic differentiation of valvular interstitial cells through RAGE/MAPK.

OBJECTIVES: We have previously shown that oxidized low-density lipoprotein (oxLDL) promotes the osteogenic differentiation of valvular interstitial cells (VICs) by inducing endoplasmic reticulum (ER) stress. We also demonstrated the detrimental role of the receptor for advanced glycation end products (RAGE) activation and signaling in the development and progression of aortic valve (AV) calcification. Here, we test the hypothesis that oxLDL may induce the osteoblastic differentiation of VICs via RAGE. METHODS: Cultured porcine aortic VICs were used in an in vitro model. The VICs were incubated with oxLDL for analysis, with and without RAGE siRNA. RESULTS: We found that oxLDL markedly increased the expression of RAGE, induced high levels of proinflammatory cytokine production and promoted the osteoblastic differentiation and calcification of VICs. oxLDL also induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) MAPK. However, these effects were found to be markedly suppressed by siRNA silencing of RAGE. CONCLUSIONS: Our data provide evidence that RAGE mediates oxLDL-induced activation of p38 and JNK MAPK and the osteogenic differentiation of VICs.

Increased risk of gonadal malignancy and prophylactic gonadectomy: a study of 102 phenotypic female patients with Y chromosome or Y-derived sequences.

STUDY QUESTION: What is the optimal protocol of management for phenotypic female patients with Y chromosome or Y-derived sequences, in particular for adult patients? SUMMARY ANSWER: Immediate gonadectomy, long-term hormone therapy and psychological care are suggested to be the optimal management for older phenotypic female patients with Y chromosome or Y-derived sequences. WHAT IS KNOWN ALREADY: Phenotypic female patients with Y chromosome or Y-derived sequences are at increasing risk of developing gonadal tumors with age. Early diagnosis and safe guidelines of management for these patients are needed. STUDY DESIGN, SIZE, DURATION: One hundred and two phenotypic women with Y chromosome or Y-derived sequences were included in a straightforward, retrospective-observational study conducted over a period of 26 years from January 1985 to November 2010. PARTICIPANTS/MATERIALS, SETTING AND METHODS: Patients aged 16-34 years presenting to our Academic Department of Gynecology with symptoms of disorders of sex development were subjected to history taking, hormonal evaluation, conventional cytogenetic analysis, PCR, histopathology and immunohistochemistry. Features of the gonads were examined and the outcome of prophylactic gonadectomy evaluated. MAIN RESULTS AND THE ROLE OF CHANCE: Among the patients recruited in our study, 48 patients (47.1%) were diagnosed with complete/partial androgen insensitivity syndrome (CAIS/PAIS) (46XY), 33 cases (32.4%) with gonadal dysgenesis (46XY) and the remaining subjects (20.1%) with mixed gonadal dysgenesis (with sex chromosome structural abnormalities). The total incidence of malignancy was 17.6%. Seventeen patients (16.7%) had gonadoblastoma, while one patient (1.0%) with gonadal dysgenesis had dysgerminoma. Gonadoblastoma were observed in 2/21 patients with sex chromosome structural abnormalities (9.5%), 3/33 patients with gonadal dysgenesis (9.1%), 9/30 patients with CAIS (30.0%) and 3/18 patients with PAIS (16.7%). LIMITATIONS, REASONS FOR CAUTION: Selection bias in this cohort study may affect data interpretation due to the low incidence of disorders of sex development in the general population. WIDER IMPLICATIONS OF THE FINDINGS: The risk for malignant transformation may occur in early life and highly increase with age in patients with Y chromosome or Y-derived sequences. Optimal timing of gonadectomy should be decided by multiple factors including the subgroup of disorder, age and degree of patient's maturity. In addition, gonadal biopsy is suggested when the disease is diagnosed and any evidence of premalignancy warranties gonadectomy. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Scientific Research Project (2013CB967404), Natural Science Funds of Zhejiang Province (Y13H04005), Zhejiang Qianjiang talent plan (2013R10027), the Fundamental Research Funds for the Central Universities and Key Projects in the National Science & Technology Pillar Program during the Eleventh Five-Year Plan Period (2012BAI32B04). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER None.

Endoplasmic reticulum stress participates in aortic valve calcification in hypercholesterolemic animals.

OBJECTIVES: Aortic valve (AV) calcification occurs via a pathophysiological process that includes lipoprotein deposition, inflammation, and osteoblastic differentiation of valvular interstitial cells. Here, we investigated the association between endoplasmic reticulum (ER) stress and AV calcification. APPROACH AND RESULTS: We identified ER stress activation in AV of patients with calcified AV stenosis. We generated an AV calcification model in hypercholesterolemic rabbits and mice, respectively, and found marked AV ER stress induction. Classical ER stress inhibitor, tauroursodeoxycholic acid, administration markedly prevented AV calcification, and attenuated AV osteoblastic differentiation and inflammation in both rabbit and mouse models of AV calcification via inhibition of ER stress. In cultured valvular interstitial cells (VICs), we found that oxidized low density lipoprotein (oxLDL) caused ER stress in a cytosolic [Ca](2+)i-dependent manner. OxLDL promoted osteoblastic differentiation via ER stress-mediated protein kinase-like ER kinase/activating transcription factor 4/osteocalcin and inositol-requiring transmembrane kinase and endonuclease-1α (IRE1α)/spliced X-box-binding protein 1/Runx2 pathway, and induced inflammatory responses through IRE1α/c-Jun N-terminal kinase and IRE1α/nuclear factor kappa-light-chain-enhancer of activated B cells signaling in VICs. Inhibition of ER stress by either tauroursodeoxycholic acid or 4-phenyl butyric acid could both suppress oxLDL-induced osteoblastic differentiation and inflammatory responses in VICs. CONCLUSIONS: These data provide novel evidence that ER stress participates in AV calcification development, and suggest that ER stress may be a novel target for AV calcification prevention and treatment.

Catheter ablation of atrial fibrillation in a patient with interruption of the inferior vena cava complicated with persistent left superior vena cava.

A 55-year-old woman of I-IVC complicated with PLSVC underwent catheter ablation for atrial fibrillation through right jugular vein access. TSP was achieved by electrocautery and the J-tip guidewire with the help of deflectable sheath and ICE. After PVI, the CS-PLSVC and LA-PLSVC connections were ablated within PLSVC.