Aggf1 Specifies Hemangioblasts at the Top of Regulatory Hierarchy via Npas4l and mTOR-S6K-Emp2-ERK Signaling.

BACKGROUND: Hemangioblasts are mesoderm-derived multipotent stem cells for differentiation of all hematopoietic and endothelial cells in the circulation system. However, the underlying molecular mechanism is poorly understood. METHODS: CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (type II CRISPR RNA-guided endonuclease) editing was used to develop aggf1-/- and emp2-/- knockout zebra fish. Whole-mount in situ hybridization and transgenic Tg(gata1-EGFP [enhanced green fluorescent protein]), Tg(mpx-EGFP), Tg(rag2-DsRed [discosoma sp. red fluorescent protein]), Tg(cd41-EGFP), Tg(kdrl-EGFP), and Tg(aggf1-/-;kdrl-EGFP) zebra fish were used to examine specification of hemangioblasts and hematopoietic stem and progenitor cells (HSPCs), hematopoiesis, and vascular development. Quantitative real-time polymerase chain reaction and Western blot analyses were used for expression analysis of genes and proteins. RESULTS: Knockout of aggf1 impaired specification of hemangioblasts and HSPCs, hematopoiesis, and vascular development in zebra fish. Expression of npas4l/cloche-the presumed earliest marker for hemangioblast specification-was significantly reduced in aggf1-/- embryos and increased by overexpression of aggf1 in embryos. Overexpression of npas4l rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels in aggf1-/- embryos, placing aggf1 upstream of npas4l in hemangioblast specification. To identify the underlying molecular mechanism, we identified emp2 as a key aggf1 downstream gene. Similar to aggf1, emp2 knockout impaired the specification of hemangioblasts and HSPCs, hematopoiesis, and angiogenesis by increasing the phosphorylation of ERK1/2 (extracellular signal-regulated protein kinase 1/2). Mechanistic studies showed that aggf1 knockdown and knockout significantly decreased the phosphorylated levels of mTOR (mammalian target of rapamycin) and p70 S6K (ribosomal protein S6 kinase), resulting in reduced protein synthesis of Emp2 (epithelial membrane protein 2), whereas mTOR activator MHY1485 (4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine) rescued the impaired specification of hemangioblasts and HSPCs and development of hematopoiesis and intersegmental vessels and reduced Emp2 expression induced by aggf1 knockdown. CONCLUSIONS: These results indicate that aggf1 acts at the top of npas4l and becomes the earliest marker during specification of hemangioblasts. Our data identify a novel signaling axis of Aggf1 (angiogenic factor with G-patch and FHA domain 1)-mTOR-S6K-ERK1/2 for specification of hemangioblasts and HSPCs, primitive and definitive hematopoiesis, and vascular development. Our findings provide important insights into specification of hemangioblasts and HSPCs essential for the development of the circulation system.

AGGF1 therapy inhibits thoracic aortic aneurysms by enhancing integrin α7-mediated inhibition of TGF-ß1 maturation and ERK1/2 signaling.

Thoracic aortic aneurysm (TAA) is a localized or diffuse dilatation of the thoracic aortas, and causes many sudden deaths each year worldwide. However, there is no effective pharmacologic therapy. Here, we show that AGGF1 effectively blocks TAA-associated arterial inflammation and remodeling in three different mouse models (mice with transverse aortic constriction, Fbn1C1041G/+ mice, and ß-aminopropionitrile-treated mice). AGGF1 expression is reduced in the ascending aortas from the three models and human TAA patients. Aggf1+/- mice and vascular smooth muscle cell (VSMC)-specific Aggf1smcKO knockout mice show aggravated TAA phenotypes. Mechanistically, AGGF1 enhances the interaction between its receptor integrin α7 and latency-associated peptide (LAP)-TGF-ß1, blocks the cleavage of LAP-TGF-ß1 to form mature TGF-ß1, and inhibits Smad2/3 and ERK1/2 phosphorylation in VSMCs. Pirfenidone, a treatment agent for idiopathic pulmonary fibrosis, inhibits TAA-associated vascular inflammation and remodeling in wild type mice, but not in Aggf1+/- mice. In conclusion, we identify an innovative AGGF1 protein therapeutic strategy to block TAA-associated vascular inflammation and remodeling, and show that efficacy of TGF-ß inhibition therapies require AGGF1.

Protein therapy of skeletal muscle atrophy and mechanism by angiogenic factor AGGF1.

BACKGROUND: Skeletal muscle atrophy is a common condition without a pharmacologic therapy. AGGF1 encodes an angiogenic factor that regulates cell differentiation, proliferation, migration, apoptosis, autophagy and endoplasmic reticulum stress, promotes vasculogenesis and angiogenesis and successfully treats cardiovascular diseases. Here, we report the important role of AGGF1 in the pathogenesis of skeletal muscle atrophy and attenuation of muscle atrophy by AGGF1. METHODS: In vivo studies were carried out in impaired leg muscles from patients with lumbar disc herniation, two mouse models for skeletal muscle atrophy (denervation and cancer cachexia) and heterozygous Aggf1+/- mice. Mouse muscle atrophy phenotypes were characterized by body weight and myotube cross-sectional areas (CSA) using H&E staining and immunostaining for dystrophin. Molecular mechanistic studies include co-immunoprecipitation (Co-IP), western blotting, quantitative real-time PCR analysis and immunostaining analysis. RESULTS: Heterozygous Aggf1+/- mice showed exacerbated phenotypes of reduced muscle mass, myotube CSA, MyHC (myosin heavy chain) and α-actin, increased inflammation (macrophage infiltration), apoptosis and fibrosis after denervation and cachexia. Intramuscular and intraperitoneal injection of recombinant AGGF1 protein attenuates atrophy phenotypes in mice with denervation (gastrocnemius weight 81.3 ± 5.7 mg vs. 67.3 ± 5.1 mg for AGGF1 vs. buffer; P < 0.05) and cachexia (133.7 ± 4.7 vs. 124.3 ± 3.2; P < 0.05). AGGF1 expression undergoes remodelling and is up-regulated in gastrocnemius and soleus muscles from atrophy mice and impaired leg muscles from patients with lumbar disc herniation by 50-60% (P < 0.01). Mechanistically, AGGF1 interacts with TWEAK (tumour necrosis factor-like weak inducer of apoptosis), which reduces interaction between TWEAK and its receptor Fn14 (fibroblast growth factor-inducing protein 14). This leads to inhibition of Fn14-induced NF-kappa B (NF-κB) p65 phosphorylation, which reduces expression of muscle-specific E3 ubiquitin ligase MuRF1 (muscle RING finger 1), resulting in increased MyHC and α-actin and partial reversal of atrophy phenotypes. Autophagy is reduced in Aggf1+/- mice due to inhibition of JNK (c-Jun N-terminal kinase) activation in denervated and cachectic muscles, and AGGF1 treatment enhances autophagy in two atrophy models by activating JNK. In impaired leg muscles of patients with lumbar disc herniation, MuRF1 is up-regulated and MyHC and α-actin are down-regulated; these effects are reversed by AGGF1 by 50% (P < 0.01). CONCLUSIONS: These results indicate that AGGF1 is a novel regulator for the pathogenesis of skeletal muscle atrophy and attenuates skeletal muscle atrophy by promoting autophagy and inhibiting MuRF1 expression through a molecular signalling pathway of AGGF1-TWEAK/Fn14-NF-κB. More importantly, the results indicate that AGGF1 protein therapy may be a novel approach to treat patients with skeletal muscle atrophy.

NINJ2 deficiency inhibits preadipocyte differentiation and promotes insulin resistance through regulating insulin signaling.

OBJECTIVE: Genetic variants in ninjurin-2 (NINJ2; nerve injury-induced protein 2) confer risk of ischemic strokes and coronary artery disease as well as endothelial activation and inflammation. However, little is known about NINJ2's in vivo functions and underlying mechanisms. METHODS: The phenotypes of NINJ2 knockout mice were analyzed, and mechanisms of NINJ2 that regulate body weight, insulin resistance, and glucose homeostasis and lipogenesis were investigated in vivo and in vitro. RESULTS: This study found that mice lacking NINJ2 showed impaired adipogenesis, increased insulin resistance, and abnormal glucose homeostasis, all of which are risk factors for strokes and coronary artery disease. Mechanistically, NINJ2 directly interacts with insulin receptor/insulin-like growth factor 1 receptor (INSR/IGF1R), and NINJ2 knockdown can block insulin-induced mitotic clonal expansion during preadipocyte differentiation by inhibiting protein kinase B/extracellular signal-regulated kinase (AKT/ERK) signaling and by decreasing the expression of key adipocyte transcriptional regulators CCAAT/enhancer-binding protein ß (C/EBP-ß), C/EBP-α, and peroxisome proliferator-activated receptor γ (PPAR-γ). Furthermore, the interaction between NINJ2 and INSR/IGF1R is needed for maintaining insulin sensitivity in adipocytes and muscle via AKT and glucose transporter type 4. Notably, adenovirus-mediated NINJ2 overexpression can ameliorate diet-induced insulin resistance in mice. CONCLUSIONS: In conclusion, these findings reveal NINJ2 as an important new facilitator of insulin receptors, and the authors propose a unique regulatory mechanism between insulin signaling, adipogenesis, and insulin resistance.

Hepatocyte Ninjurin2 promotes hepatic stellate cell activation and liver fibrosis through the IGF1R/EGR1/PDGF-BB signaling pathway.

BACKGROUND: Liver fibrogenesis is orchestrated by the paracrine signaling interaction between several resident cell types regulating the activation of hepatic stellate cells (HSCs). However, the molecular mechanisms underlying paracrine regulation are largely unknown. The aim of this study is to elucidate the role of Ninjurin2 in the crosstalk between hepatocytes and HSCs and better understand the implications of Ninjurin2 in liver fibrosis. METHODS: Ninj2 knockout mice (Ninj2-/-) and hepatocyte-specific Ninj2 overexpression mice (Ninj2Hep-tg) were constructed and followed by the induction of liver fibrosis using methionine- and choline-deficient (MCD) diet. The relationship between Ninjurin2 and liver fibrosis phenotype was evaluated in vivo by measurement of fibrotic markers and related genes. We used an in vitro transwell cell co-culture model to examine the impact of Ninjurin2 in hepatocytes on the crosstalk to HSCs. The interaction of Ninjurin2 and IGF1R and the regulation of PI3K-AKT-EGR1 were analyzed in vivo and in vitro. Finally, an inhibitory Ninjurin2 peptide was injected intravenously via the tail vein to investigate whether inhibiting of Ninjurin2 cascade can attenuate MCD diet-induced liver fibrosis in mice. RESULTS: We found that hepatic Ninjurin2 expression was significantly increased in fibrotic human liver and MCD diet-induced liver injury mouse models. In the mouse model, hepatocyte-specific overexpression of Ninj2 exacerbates MCD-induced liver fibrosis, while global Ninj2 knockout reverses the phenotype. To mimic hepatocyte-HSC crosstalk during liver fibrosis, we used co-culture systems containing hepatocytes and HSCs and determined that Ninjurin2 overexpression in hepatocytes directly activates HSCs in vitro. Mechanistically, Ninjurin2 directly interacts with insulin-like growth factor 1 receptor (IGF1R) and increases the hepatocyte secretion of the fibrogenic cytokine, platelet-derived growth factor-BB (PDGF-BB) through IGF1R-PI3K-AKT-EGR1 cascade. Inhibition of PDGFRB signaling in HSCs can abolish the profibrogenic effect of Ninjurin2. In addition, we demonstrated that a specific inhibitory Ninjurin2 peptide containing an N-terminal adhesion motif mitigates liver fibrosis and improves hepatic function in the mouse models by negatively regulating the sensitivity of IGF1R to IGF1 in hepatocytes. CONCLUSION: Hepatic Ninjurin2 plays a key role in liver fibrosis through paracrine regulation of PDGF-BB/PDGFRB signaling in HSCs, and the results suggesting Ninjurin2 may be a potential therapeutic target.

Two Novel Functional Mutations in Promoter Region of SCN3B Gene Associated with Atrial Fibrillation.

The sodium voltage-gated channel beta subunit 3 (SCN3B) plays a crucial role in electrically excitable cells and conduction tissue in the heart. Some previous studies have established that genetic modification in sodium voltage-channel genes encoding for the cardiac ß-subunits, such as SCN1B, SCN2B, SCN3B and SCN4B, can result in atrial fibrillation (AF). In the current study, we identified two rare variants in 5'UTR (NM_018400.4: c.-324C>A, rs976125894 and NM_018400.4: c.-303C>T, rs1284768362) of SCN3B in two unrelated lone AF patients. Our further functional studies discovered that one of them, the A allele of c.-324C>A (rs976125894), can improve transcriptional activity and may raise SCN3B expression levels. The A allele of c.-324C>A (rs976125894) has higher transcriptional activity when it interacts with GATA4, as we confirmed transcription factor GATA4 is a regulator of SCN3B. To the best of our knowledge, the current study is the first to demonstrate that the gain-of-function mutation of SCN3B can produce AF and the first to link a mutation occurring in the non-coding 5'UTR region of SCN3B to lone AF. The work also offers empirical proof that GATA4 is a critical regulator of SCN3B gene regulation. Our findings may serve as an encyclopedia for AF susceptibility variants and can also provide insight into the investigation of the functional mechanisms behind AF variants discovered by genetic methods.

Genome-Wide Association Study for Idiopathic Ventricular Tachyarrhythmias Identifies Key Role of CCR7 and PKN2 in Calcium Homeostasis and Cardiac Rhythm Maintenance.

BACKGROUND: Idiopathic ventricular tachycardia (VT) occurs in structurally normal hearts and accounts for a significant number of all types of VT. The genome-wide association study is the most effective strategy for identifying novel genetic variants for common diseases. However, no genome-wide association study has been reported for idiopathic VT. METHODS: We conducted the first genome-wide association study for idiopathic VT in the Chinese Han population using a discovery population with 246 cases and 648 controls and a replication population with 222 cases and >4072 controls. Candidate VT genes were functionally characterized in zebrafish. Real-time RT-PCR analysis was used to determine the effects of candidate genes on expression of ion channels and regulators. Patch-clamping was used to record L-type calcium current from neonatal rat cardiomyocytes with overexpression of candidate genes. RESULTS: We identified 4 significant loci represented by rs78960694 (minor allele frequency [MAF]=5.02% in cases and 1.84% in controls; P=4.30×10-12, odds ratio [OR]=3.91) and rs2229095 (MAF=3.25% in cases and 1.63% in controls; P=1.02×10-7, OR=3.44) near and in CCR7, respectively, rs68126098 in NELL1 (MAF=40.98% in cases and 32.07% in controls; P=2.40×10-8, OR=1.53), rs2390325 between PKN2 and LMO4 (MAF=21.19% in cases and 15.12% in controls; P=1.92×10-7, OR=1.62), and rs270065 in CSMD1 (MAF=33.63% in cases and 40.25% in controls; P=9.51×10-7, OR=0.69). Note that the associations of idiopathic VT for CCR7 variant rs78960694 and NELL1 variant rs68126098 reach genome-wide significance (P<5.00×10-8). Overexpression of either PKN2 or CCR7 increased the heart rate in zebrafish, and enhanced expression of CACNA1C, RYR2, or NOS1AP in zebrafish embryos, HEK293, and AC16 cardiomyocytes. Overexpression of either PKN2 or CCR7 significantly increased L-type Ca2+ current density. CONCLUSIONS: The first genome-wide association study identifies 4 novel loci and 2 risk genes (PKN2 and CCR7) for idiopathic VT. These findings identify new molecular determinants for cardiac calcium homeostasis and rhythm maintenance and provide novel targets for diagnosis and treatment for idiopathic VT.

Gene therapy targeting protein trafficking regulator MOG1 in mouse models of Brugada syndrome, arrhythmias, and mild cardiomyopathy.

Brugada syndrome (BrS) is a fatal arrhythmia that causes an estimated 4% of all sudden death in high-incidence areas. SCN5A encodes cardiac sodium channel NaV1.5 and causes 25 to 30% of BrS cases. Here, we report generation of a knock-in (KI) mouse model of BrS (Scn5aG1746R/+). Heterozygous KI mice recapitulated some of the clinical features of BrS, including an ST segment abnormality (a prominent J wave) on electrocardiograms and development of spontaneous ventricular tachyarrhythmias (VTs), seizures, and sudden death. VTs were caused by shortened cardiac action potential duration and late phase 3 early afterdepolarizations associated with reduced sodium current density (INa) and increased Kcnd3 and Cacna1c expression. We developed a gene therapy using adeno-associated virus serotype 9 (AAV9) vector-mediated MOG1 delivery for up-regulation of MOG1, a chaperone that binds to NaV1.5 and traffics it to the cell surface. MOG1 was chosen for gene therapy because the large size of the SCN5A coding sequence (6048 base pairs) exceeds the packaging capacity of AAV vectors. AAV9-MOG1 gene therapy increased cell surface expression of NaV1.5 and ventricular INa, reversed up-regulation of Kcnd3 and Cacna1c expression, normalized cardiac action potential abnormalities, abolished J waves, and blocked VT in Scn5aG1746R/+ mice. Gene therapy also rescued the phenotypes of cardiac arrhythmias and contractile dysfunction in heterozygous humanized KI mice with SCN5A mutation p.D1275N. Using a small chaperone protein may have broad implications for targeting disease-causing genes exceeding the size capacity of AAV vectors.

Mog1 deficiency promotes cardiac contractile dysfunction and isoproterenol-induced arrhythmias associated with cardiac fibrosis and Cx43 remodeling.

Our earlier studies identified MOG1 as a Nav1.5-binding protein that promotes Nav1.5 intracellular trafficking to plasma membranes. Genetic studies have identified MOG1 variants responsible for cardiac arrhythmias. However, the physiological functions of MOG1 in vivo remain incompletely characterized. In this study, we generated Mog1 knockout (Mog1-/-) mice. Mog1-/- mice did not develop spontaneous arrhythmias at the baseline, but exhibited a prolongation of QRS duration. Mog1-/- mice treated with isoproterenol (ISO), but not with flecainide, exhibited an increased risk of arrhythmias and even sudden death. Mog1-/- mice had normal cardiac morphology, however, LV systolic dysfunction was identified and associated with an increase in ventricular fibrosis. Whole-cell patch-clamping and Western blotting analysis clearly demonstrated the normal cardiac expression and function of Nav1.5 in Mog1-/- mice. Further RNA-seq and iTRAQ analysis identified critical pathways and genes, including extracellular matrix (Mmp2), gap junction (Gja1), and mitochondrial components that were dysregulated in Mog1-/- mice. RT-qPCR, Western blotting, and immunofluorescence assays revealed reduced cardiac expression of Gja1 in Mog1-/- mice. Dye transfer assays confirmed impairment of gap-junction function; Cx43 gap-junction enhancer ZP123 decreased arrhythmia inducibility in ISO-treated Mog1-/- mice. Transmission electron microscopy analysis revealed abnormal sarcomere ultrastructure and altered mitochondrial morphology in Mog1-/- mice. Mitochondrial dynamics was found to be disturbed, and associated with a trend toward increased mitochondrial fusion in Mog1-/- mice. Meanwhile, the level of ATP supply was increased in the hearts of Mog1-/- mice. These results indicate that MOG1 plays an important role in cardiac electrophysiology and cardiac contractile function.

Identification and characterization of a special type of subnuclear structure: AGGF1-coated paraspeckles.

AGGF1 is an angiogenic factor with G-Patch and FHA domains 1 described by our group. Gain-of-function mutations in AGGF1 cause Klippel-Trenaunay syndrome, whereas somatic loss-of-function mutations cause cancer. Paraspeckles are small membraneless subnuclear structures with a diameter of 0.5-1 µm, and composed of lncRNA NEAT1 as the scaffold and three core RNA-binding proteins NONO, PSPC1, and PSF. Here, we show that AGGF1 is a key regulatory and structural component of paraspeckles that induces paraspeckle formation, forms an outside rim of paraspeckles, wraps around the NONO/PSF/PSPC1/NEAT1 core, and regulates the size and number of paraspeckles. AGGF1-paraspeckles are larger (>1 µm) than conventional paraspeckles. RNA-FISH in combination with immunostaining shows that AGGF1, NONO, and NEAT1_2 co-localize in 20.58% of NEAT1_2-positive paraspeckles. Mechanistically, AGGF1 interacts with NONO, PSF, and HNRNPK, and upregulates NEAT1_2, a longer, 23 kb NEAT1 transcript with a key role in regulation of paraspeckle size and number. RNA-immunoprecipitation shows that AGGF1 interacts with NEAT1, which may be another possible mechanism underlying the formation of AGGF1-paraspeckles. NEAT1_2 knockdown reduces the number and size of AGGF1-paraspeckles. Functionally, AGGF1 regulates alternative RNA splicing as it decreases the exon skipping/inclusion ratio in a CD44 model. AGGF1 is also localized in some nuclear foci without NEAT1 or NONO, suggesting that AGGF1 is an important liquid-liquid phase separation (LLPS) driver for other types of AGGF1-positive nuclear condensates (referred to as AGGF1-bodies). Our results identify a special type of AGGF1-coated paraspeckles and provide important insights into the formation, structure, and function of paraspeckles.

Hyperlipidemia patients carrying LDLR splicing mutation c.1187-2A>G respond favorably to rosuvastatin and PCSK9 inhibitor evolocumab.

Mutations in the LDL receptor gene LDLR cause familial hypercholesterolemia (FH); however, the pharmacogenomics of specific LDLR mutations remains poorly understood. The goals of this study were to identify the genetic cause of a three-generation Chinese family affected with autosomal dominant FH, and to investigate the response of FH patients in the family to statin and evolocumab. Whole exome sequencing of the FH family with four patients and six unaffected members identified a heterozygous splicing mutation (c.1187-2A>G) in LDLR. The mutation co-segregated with FH in the family, providing strong genetic evidence to support its pathogenicity. The proband was a 48-year-old male FH patient who had an acute myocardial infarction (MI) and ventricular fibrillation (VF), and showed LDL-C of 5.23 mmol/L. A combination of life style modifications on food and exercise and treatment with rosuvastatin reduced his LDL-C to 2.05-2.80 mmol/L. Addition of ezetimibe did not improve rosuvastatin therapy, but addition of evolocumab further reduced LDL-C by 70% to 0.7 mmol/L at the first time and by 67% to 1.31 mmol/L at the second time. Rosuvastatin also reduced LDL-C for proband's father and sister by 40% and 43-63%, respectively. Lovastatin alone or addition to rosuvastatin treatment did not have any effect on LDL-C for the proband and his son. Both patients carry ApoE 3/4 genotype and SLCO1B1 rs4149056 TT genotype. These results suggest that combined treatment with rosuvastatin (but not lovastatin or ezetimibe) and evolocumab can control LDL-C to meet the LDL-C treatment goal for patients with LDLR splicing mutation c.1187-2A>G.

Angiogenic factor AGGF1 blocks neointimal formation after vascular injury via interaction with integrin α7 on vascular smooth muscle cells.

Angiogenic factor AGGF1 (AngioGenic factor with G-patch and FHA (Forkhead-Associated) domain 1) blocks neointimal formation (formation of a new or thickened layer of arterial intima) after vascular injury by regulating phenotypic switching of vascular smooth muscle cells (VSMCs). However, the AGGF1 receptor on VSMCs and the underlying molecular mechanisms of its action are unknown. In this study, we used functional analysis of serial AGGF1 deletions to reveal the critical AGGF1 domain involved in VSMC phenotypic switching. This domain was required for VSMC phenotypic switching, proliferation, cell cycle regulation, and migration, as well as the regulation of cell cycle inhibitors cyclin D, p27, and p21. This domain also contains an RDDAPAS motif via which AGGF1 interacts with integrin α7 (ITGA7), but not α8. In addition, we show that AGGF1 enhanced the expression of contractile markers MYH11, α-SMA, and SM22 and inhibited MEK1/2, ERK1/2, and ELK phosphorylation in VSMCs, and that these effects were inhibited by knockdown of ITGA7, but not by knockdown of ITGA8. In vivo, deletion of the VSMC phenotypic switching domain in mice with vascular injury inhibited the functions of AGGF1 in upregulating α-SMA and SM22, inhibiting MEK1/2, ERK1/2, and ELK phosphorylation, in VSMC proliferation, and in blocking neointimal formation. Finally, we show the inhibitory effect of AGGF1 on neointimal formation was blocked by lentivirus-delivered shRNA targeting ITGA7. Our data demonstrate that AGGF1 interacts with its receptor integrin α7 on VSMCs, and this interaction is required for AGGF1 signaling in VSMCs and for attenuation of neointimal formation after vascular injury.

Identification and functional analysis of two new de novo KCNMA1 variants associated with Liang-Wang syndrome.

AIM: Loss-of-function KCNMA1 variants cause Liang-Wang syndrome (MIM #618729), a newly identified multiple malformation syndrome with a broad spectrum of developmental and neurological phenotypes. However, the full spectrum of clinical features and underlying pathogenic mechanisms need full elucidation. METHODS: Exome sequencing was used to identify pathogenic variants. Patch-clamp recordings were performed to access the effects of KCNMA1 variants on BK channels. Total and membrane protein expression levels of BK channels were characterized using Western blotting. RESULTS: We report identification and functional characterization of two new de novo loss-of-function KCNMA1 variants p.(A172T) and p.(A314T) with characteristics of Liang-Wang syndrome. Variant p.(A172T) is associated with developmental delay, cognitive impairment and ataxia. Mechanistically, p.(A172T) abolishes BK potassium current, inhibits Mg2+ -dependent gating, but shifts conductance-voltage (G-V) curves to more positive potentials when complexed with WT channels. Variant p.(A314T) is associated with developmental delay, intellectual disability, cognitive impairment, mild ataxia and generalized epilepsy; suppresses BK current amplitude; and shifts G-V curves to more positive potentials when expressed with WT channels. In addition, two new patients with previously reported gain-of-function variants p.(N536H) and p.(N995S) are found to show epilepsy and paroxysmal dyskinesia as reported previously, but also exhibit additional symptoms of cognitive impairment and dysmorphic features. Furthermore, variants p.(A314T) and p.(N536H) reduced total and membrane levels of BK proteins. CONCLUSION: Our findings identified two new loss-of-function mutations of KCNMA1 associated with Liang-Wang syndrome, expanded the spectrum of clinical features associated with gain-of-function KCNMA1 variants and emphasized the overlapping features shared by gain-of-function and loss-of-function mutations.

Mechanistic insights into the interaction of cardiac sodium channel Nav1.5 with MOG1 and a new molecular mechanism for Brugada syndrome.

BACKGROUND: Mutations in cardiac sodium channel Nav1.5 cause Brugada syndrome (BrS). MOG1 is a chaperone that binds to Nav1.5, facilitates Nav1.5 trafficking to the cell surface, and enhances the amplitude of sodium current INa. OBJECTIVE: The purpose of this study was to identify structural elements involved in MOG1-Nav1.5 interaction and their relevance to the pathogenesis of BrS. METHODS: Systematic analyses of large deletions, microdeletions, and point mutations, and glutathione S-transferases pull-down, co-immunoprecipitation, cell surface protein quantification, and patch-clamping of INa were performed. RESULTS: Large deletion analysis defined the MOG1-Nav1.5 interaction domain to amino acids S476-H585 of Nav1.5 Loop I connecting transmembrane domains I and II. Microdeletion and point mutation analyses further defined the domain to F530T531F532R533R534R535. Mutations F530A, F532A, R533A, and R534A, but not T531A and R535A, significantly reduced MOG1-Nav1.5 interaction and eliminated MOG1-enhanced INa. Mutagenesis analysis identified D24, E36, D44, E53, and E101A of MOG1 as critical residues for interaction with Nav1.5 Loop I. We then characterized 3 mutations at the MOG1-Nav1.5 interaction domain: p.F530V, p.F532C, and p.R535Q reported from patients with long QT syndrome and BrS. We found that p.F532C reduced MOG1-Nav1.5 interaction and eliminated MOG1 function on INa; p.R535Q is also a loss-of-function mutation that reduces INa amplitude in a MOG1-independent manner, whereas p.F530V is benign as it does not have an apparent effect on MOG1 and INa. CONCLUSION: Our findings define the MOG1-Nav1.5 interaction domain to a 5-amino-acid motif of F530T531F532R533R534 in Loop I. Mutation p.F532C associated with BrS abolishes Nav1.5 interaction with MOG1 and reduces MOG1-enhanced INa density, thereby uncovering a novel molecular mechanism for the pathogenesis of BrS.

Endothelial cell metabolic memory causes cardiovascular dysfunction in diabetes.

AIMS: The aim of this study was to identify the molecular mechanism for hyperglycaemia-induced metabolic memory in endothelial cells (ECs), and to show its critical importance to development of cardiovascular dysfunction in diabetes. METHODS AND RESULTS: Hyperglycaemia induces increased nuclear factor-κB (NF-κB) signalling, up-regulation of miR-27a-3p, down-regulation of nuclear factor erythroid-2 related factor 2 (NRF2) expression, increased transforming growth factor-ß (TGF-ß) signalling, down-regulation of miR-29, and induction of endothelial-to-mesenchymal transition (EndMT), all of which are memorized by ECs and not erased when switched to a low glucose condition, thereby causing perivascular fibrosis and cardiac dysfunction. Similar metabolic memory effects are found for production of nitric oxide (NO), generation of reactive oxygen species (ROS), and the mitochondrial oxygen consumption rate in two different types of ECs. The observed metabolic memory effects in ECs are blocked by NRF2 activator tert-butylhydroquinone and a miR-27a-3p inhibitor. In vivo, the NRF2 activator and miR-27a-3p inhibitor block cardiac perivascular fibrosis and restore cardiovascular function by decreasing NF-κB signalling, down-regulating miR-27a-3p, up-regulating NRF2 expression, reducing TGF-ß signalling, and inhibiting EndMT during insulin treatment of diabetes in streptozotocin-induced diabetic mice, whereas insulin alone does not improve cardiac function. CONCLUSIONS: Our data indicate that disruption of hyperglycaemia-induced EC metabolic memory is required for restoring cardiac function during treatment of diabetes, and identify a novel molecular signalling pathway of NF-κB/miR-27a-3p/NRF2/ROS/TGF-ß/EndMT involved in metabolic memory.

Genetic association analysis between IL9 and coronary artery disease in a Chinese Han population.

Interleukin-9 (IL-9) plays important role in coronary artery disease (CAD). However, the exact relationship between them is not explored yet. Here, four tag SNPs covering IL9 (rs31563, rs2069868, rs2069870 and rs31564) were selected to conduct case-control association analyses in a total of 3704 individuals from Chinese Han population (1863 CAD vs 1841 control). Results showed that: first, rs2069868 was associated with CAD combined with hypertension (Padj = 0.027); second, IL9 haplotype (CGAT) was associated with CAD (Padj = 0.035), and the combination genotype of "rs31563_CC/rs31564_TT" would remarkably decrease the risk of CAD (Padj = 0.001); third, significant associations were found between rs2069870 and decreased LDL-c levels and decreased total cholesterol levels, and between rs31563 and increased HDL-c levels (Padj < 0.05). Therefore, we conclude that IL9 might play a causal role in CAD by interacted with CAD traditional risk factors, which might confer a new way to improve the prevention and treatment of CAD.

Functional rare variant in a C/EBPbeta binding site in NINJ2 gene increases the risk of coronary artery disease.

OBJECTIVE: NINJ2 regulates activation of vascular endothelial cells, and genome-wide association studies showed that variants in NINJ2 confer risk to stroke. However, whether variants in NINJ2 are associated with coronary artery disease (CAD) is unknown. METHODS: We genotyped rs34166160 in NINJ2 in two independent Chinese GeneID populations which included 2,794 CAD cases and 4,131 controls, and performed genetics association studies. Functional studies were also performed to reveal the mechanisms. RESULTS: Allele rs34166160 significantly confers risk to CAD in the GeneID Hubei population which contained 1,440 CAD cases and 2,660 CAD-free controls (observed P-obs = 6.39 × 10-3 with an odds ratio (OR) was 3.39, adjusted P-adj = 8.12 × 10-3 with an OR of 3.10). The association was replicated in another population, GeneID Shandong population contained 1,354 CAD cases and 1,471 controls (P-obs = 3.33 × 10-3 with an OR of 3.14, P-adj = 0.01 with an OR of 2.74). After combining the two populations, the association was more significant (P-obs = 1.57 × 10-5 with an OR of 3.58, P-adj = 3.41 × 10-4 with an OR of 2.80). In addition, we found that rs34166160 was associated with the mRNA expression level of NINJ2 and the flanking region of rs34166160 can directly bind with transcriptional factor CCAAT-box/enhancer-binding protein beta, and the risk A allele has more transcription activity than non-risk C allele with or without LPS in HUVEC cells. CONCLUSIONS: Our study demonstrates that the functional rare variant rs34166160 in NINJ2 confers risk to CAD for the first time, and these findings further expand the range of the pathology of CAD and atherosclerosis.

Role of epigenetic m6 A RNA methylation in vascular development: mettl3 regulates vascular development through PHLPP2/mTOR-AKT signaling.

N6 -methyladenosine (m6A) methylation is the most prevalent RNA modification, and it emerges as an important regulatory mechanism of gene expression involved in many cellular and biological processes. However, the role of m6 A methylation in vascular development is not clear. The m6 A RNA methylation is regulated by dynamic interplay among methyltransferases, binding proteins, and demethylases. Mettl3 is a member of the mettl3-mettl14 methyltransferase complex, referred to as writers that catalyze m6A RNA methylation. Here, we used CRISPR-Cas9 genome editing to develop two lines of knockout (KO) zebrafish for mettl3. Heterozygous mettl3+/- KO embryos show defective vascular development, which is directly visible in fli-EGFP and flk-EGFP zebrafish. Alkaline phosphatase staining and whole mount in situ hybridization with cdh5, and flk markers demonstrated defective development of intersegmental vessels (ISVs), subintestinal vessels (SIVs), interconnecting vessels (ICVs) and dorsal longitudinal anastomotic vessels (DLAV) in both heterozygous mettl3+/- and homozygous mettl3-/- KO zebrafish embryos. Similar phenotypes were observed in zebrafish embryos with morpholino knockdown (KD) of mettl3; however, the vascular defects were rescued fully by overexpression of constitutively active AKT1. KD of METTL3 in human endothelial cells inhibited cell proliferation, migration, and capillary tube formation. Mechanistically, mettl3 KO and KD significantly reduced the levels of m6 A RNA methylation, and AKT phosphorylation (S473) by an increase in the expression of phosphatase enzyme PHLPP2 and reduction in the phosphorylation of mTOR (S2481), a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases. These data suggest that m6 A RNA methylation regulates vascular development via PHLPP2/mTOR-AKT signaling.

TAGAP instructs Th17 differentiation by bridging Dectin activation to EPHB2 signaling in innate antifungal response.

The TAGAP gene locus has been linked to several infectious diseases or autoimmune diseases, including candidemia and multiple sclerosis. While previous studies have described a role of TAGAP in T cells, much less is known about its function in other cell types. Here we report that TAGAP is required for Dectin-induced anti-fungal signaling and proinflammatory cytokine production in myeloid cells. Following stimulation with Dectin ligands, TAGAP is phosphorylated by EPHB2 at tyrosine 310, which bridges proximal Dectin-induced EPHB2 activity to downstream CARD9-mediated signaling pathways. During Candida albicans infection, mice lacking TAGAP mount defective immune responses, impaired Th17 cell differentiation, and higher fungal burden. Similarly, in experimental autoimmune encephalomyelitis model of multiple sclerosis, TAGAP deficient mice develop significantly attenuated disease. In summary, we report that TAGAP plays an important role in linking Dectin-induced signaling to the promotion of effective T helper cell immune responses, during both anti-fungal host defense and autoimmunity.

Deficiency of SCAMP5 leads to pediatric epilepsy and dysregulation of neurotransmitter release in the brain.

Secretory carrier membrane proteins (SCAMPs) play an important role in exocytosis in animals, but the precise function of SCAMPs in human disease is unknown. In this study, we identified a homozygous mutation, SCAMP5 R91W, in a Chinese consanguineous family with pediatric epilepsy and juvenile Parkinson's disease. Scamp5 R91W mutant knock-in mice showed typical early-onset epilepsy similar to that in humans. Single-neuron electrophysiological recordings showed that the R91W mutation significantly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) at a resting state and also increased the amplitude of evoked EPSCs. The R91W mutation affected the interaction between SCAMP5 and synaptotagmin 1 and may affect the function of the SNARE complex, the machinery required for vesicular trafficking and neurotransmitter release. Our work shows that dysfunction of SCAMP5 shifted the excitation/inhibition balance of the neuronal network in the brain, and the deficiency of SCAMP5 leads to pediatric epilepsy.