AGGF1 is an angiogenic factor with therapeutic potential to treat coronary artery disease (CAD) and myocardial infarction (MI). However, the underlying mechanism for AGGF1-mediated therapeutic angiogenesis is unknown. Here, we show for the first time that AGGF1 activates autophagy, a housekeeping catabolic cellular process, in endothelial cells (ECs), HL1, H9C2, and vascular smooth muscle cells. Studies with Atg5 small interfering RNA (siRNA) and the autophagy inhibitors bafilomycin A1 (Baf) and chloroquine demonstrate that autophagy is required for AGGF1-mediated EC proliferation, migration, capillary tube formation, and aortic ring-based angiogenesis. Aggf1+/- knockout (KO) mice show reduced autophagy, which was associated with inhibition of angiogenesis, larger infarct areas, and contractile dysfunction after MI. Protein therapy with AGGF1 leads to robust recovery of myocardial function and contraction with increased survival, increased ejection fraction, reduction of infarct areas, and inhibition of cardiac apoptosis and fibrosis by promoting therapeutic angiogenesis in mice with MI. Inhibition of autophagy in mice by bafilomycin A1 or in Becn1+/- and Atg5 KO mice eliminates AGGF1-mediated angiogenesis and therapeutic actions, indicating that autophagy acts upstream of and is essential for angiogenesis. Mechanistically, AGGF1 initiates autophagy by activating JNK, which leads to activation of Vps34 lipid kinase and the assembly of Becn1-Vps34-Atg14 complex involved in the initiation of autophagy. Our data demonstrate that (1) autophagy is essential for effective therapeutic angiogenesis to treat CAD and MI; (2) AGGF1 is critical to induction of autophagy; and (3) AGGF1 is a novel agent for treatment of CAD and MI. Our data suggest that maintaining or increasing autophagy is a highly innovative strategy to robustly boost the efficacy of therapeutic angiogenesis.

Aggf1 is the first gene identified for Klippel-Trenaunay syndrome (KTS), and encodes an angiogenic factor. However, the in vivo roles of Aggf1 are incompletely defined. Here we demonstrate that Aggf1 is essential for both physiological angiogenesis and pathological tumour angiogenesis in vivo. Two lines of Aggf1 knockout (KO) mice showed a particularly severe phenotype as no homozygous embryos were observed and heterozygous mice also showed embryonic lethality (haploinsufficient lethality) observed only for Vegfa and Dll4. Aggf1+/- KO caused defective angiogenesis in yolk sacs and embryos. Survived adult heterozygous mice exhibit frequent haemorrhages and increased vascular permeability due to increased phosphorylation and reduced membrane localization of VE-cadherin. AGGF1 inhibits VE-cadherin phosphorylation, increases plasma membrane VE-cadherin in ECs and in mice, blocks vascular permeability induced by ischaemia-reperfusion (IR), restores depressed cardiac function and contraction, reduces infarct sizes, cardiac fibrosis and necrosis, haemorrhages, edema, and macrophage density associated with IR. Mechanistically, AGGF1 promotes angiogenesis by activating catalytic p110α subunit and p85α regulatory subunit of PI3K, leading to activation of AKT, GSK3β and p70S6K. AKT activation is significantly reduced in heterozygous KO mice and isolated KO ECs, which can be rescued by exogenous AGGF1. ECs from KO mice show reduced capillary angiogenesis, which is rescued by AGGF1 and AKT. Tumour growth/angiogenesis is reduced in heterozygous mice, which was associated with reduced activation of p110α, p85α and AKT. Together with recent identification of somatic mutations in p110α (encoded by PIK3CA), our data establish a potential mechanistic link between AGGF1 and PIK3CA, the two genes identified for KTS.

Atrial fibrillation (AF) is the most common cardiac arrhythmia at the clinic. Recent GWAS identified several variants associated with AF, but they account for <10% of heritability. Gene-gene interaction is assumed to account for a significant portion of missing heritability. Among GWAS loci for AF, only three were replicated in the Chinese Han population, including SNP rs2106261 (G/A substitution) in ZFHX3, rs2200733 (C/T substitution) near PITX2c, and rs3807989 (A/G substitution) in CAV1. Thus, we analyzed the interaction among these three AF loci. We demonstrated significant interaction between rs2106261 and rs2200733 in three independent populations and combined population with 2,020 cases/5,315 controls. Compared to non-risk genotype GGCC, two-locus risk genotype AATT showed the highest odds ratio in three independent populations and the combined population (OR=5.36 (95% CI 3.87-7.43), P=8.00×10-24). The OR of 5.36 for AATT was significantly higher than the combined OR of 3.31 for both GGTT and AACC, suggesting a synergistic interaction between rs2106261 and rs2200733. Relative excess risk due to interaction (RERI) analysis also revealed significant interaction between rs2106261 and rs2200733 when exposed two copies of risk alleles (RERI=2.87, P<1.00×10-4) or exposed to one additional copy of risk allele (RERI=1.29, P<1.00×10-4). The INTERSNP program identified significant genotypic interaction between rs2106261 and rs2200733 under an additive by additive model (OR=0.85, 95% CI: 0.74-0.97, P=0.02). Mechanistically, PITX2c negatively regulates expression of miR-1, which negatively regulates expression of ZFHX3, resulting in a positive regulation of ZFHX3 by PITX2c; ZFHX3 positively regulates expression of PITX2C, resulting in a cyclic loop of cross-regulation between ZFHX3 and PITX2c. Both ZFHX3 and PITX2c regulate expression of NPPA, TBX5 and NKX2.5. These results suggest that cyclic cross-regulation of gene expression is a molecular basis for gene-gene interactions involved in genetics of complex disease traits.

BackgroundTo evaluate the feasibility of using radiomics with precontrast magnetic resonance imaging for classifying hepatocellular carcinoma (HCC) and hepatic haemangioma (HH).MethodsThis study enrolled 369 consecutive patients with 446 lesions (a total of 222 HCCs and 224 HHs). A training set was constituted by randomly selecting 80% of the samples and the remaining samples were used to test. On magnetic resonance (MR) images of HCC and HH obtained with in-phase, out-phase, T2-weighted imaging (T2WI), and diffusion-weighted imaging (DWI) sequences, we outlined the target lesions and extracted 1029 radiomics features, which were classified as first-, second-, higher-order statistics and shape features. Then, the variance threshold, select k best, and least absolute shrinkage and selection operator algorithms were explored for dimensionality reduction of the features. We used four classifiers (decision tree, random forest, K nearest neighbours, and logistic regression) to identify HCC and HH on the basis of radiomics features. Two abdominal radiologists also performed the conventional qualitative analysis for classification of HCC and HH. Diagnostic performances of radiomics and radiologists were evaluated by receiver operating characteristic (ROC) analysis.ResultsValuable radiomics features for building a radiomics signature were extracted from in-phase (n = 22), out-phase (n = 24), T2WI (n = 34) and DWI (n = 24) sequences. In comparison, the logistic regression classifier showed better predictive ability by combining four sequences. In the training set, the area under the ROC curve (AUC) was 0.86 (sensitivity: 0.76; specificity: 0.78), and in the testing set, the AUC was 0.89 (sensitivity: 0.822; specificity: 0.714). The diagnostic performance for the optimal radiomics-based combined model was significantly higher than that for the less experienced radiologist (2-years experience) (AUC = 0.702, p < 0.05), and had no statistic difference with the experienced radiologist (10-years experience) (AUC = 0.908, p>0.05).ConclusionsWe developed and validated a radiomics signature as an adjunct tool to distinguish HCC and HH by combining in-phase, out-phase, T2W, and DW MR images, which outperformed the less experienced radiologist (2-years experience), and was nearly equal to the experienced radiologist (10-years experience).

Aims Cardiac fibrosis is a major cause of heart failure (HF), and mediated by the differentiation of cardiac fibroblasts into myofibroblasts. However, limited tools are available to block cardiac fibrosis. ADAMTS16 is a member of the ADAMTS superfamily of extracellular protease enzymes involved in extracellular matrix (ECM) degradation and remodelling. In this study, we aimed to establish ADAMTS16 as a key regulator of cardiac fibrosis. Methods and results Western blot and qRT–PCR analyses demonstrated that ADAMTS16 was significantly up-regulated in mice with transverse aortic constriction (TAC) associated with left ventricular hypertrophy and HF, which was correlated with increased expression of Mmp2, Mmp9, Col1a1, and Col3a1. Overexpression of ADAMTS16 accelerated the AngII-induced activation of cardiac fibroblasts into myofibroblasts. Protein structural analysis and co-immunoprecipitation revealed that ADAMTS16 interacted with the latency-associated peptide (LAP)-transforming growth factor (TGF)-β via a RRFR motif. Overexpression of ADAMTS16 induced the activation of TGF-β in cardiac fibroblasts; however, the effects were blocked by a mutation of the RRFR motif to IIFI, knockdown of Adamts16 expression, or a TGF-β-neutralizing antibody (ΝAb). The RRFR tetrapeptide, but not control IIFI peptide, blocked the interaction between ADAMTS16 and LAP-TGF-β, and accelerated the activation of TGF-β in cardiac fibroblasts. In TAC mice, the RRFR tetrapeptide aggravated cardiac fibrosis and hypertrophy by up-regulation of ECM proteins, activation of TGF-β, and increased SMAD2/SMAD3 signalling, however, the effects were blocked by TGF-β-NAb. Conclusion ADAMTS16 promotes cardiac fibrosis, cardiac hypertrophy, and HF by facilitating cardiac fibroblasts activation via interacting with and activating LAP-TGF-β signalling. The RRFR motif of ADAMTS16 disrupts the interaction between ADAMTS16 and LAP-TGF-β, activates TGF-β, and aggravated cardiac fibrosis and hypertrophy. This study identifies a novel regulator of TGF-β signalling and cardiac fibrosis, and provides a new target for the development of therapeutic treatment of cardiac fibrosis and HF.