Design and Identification of a Novel Antiviral Affinity Peptide against Fowl Adenovirus Serotype 4 (FAdV-4) by Targeting Fiber2 Protein.

Outbreaks of hydropericardium hepatitis syndrome caused by fowl adenovirus serotype 4 (FAdV-4) with a novel genotype have been reported in China since 2015, with significant economic losses to the poultry industry. Fiber2 is one of the important structural proteins on FAdV-4 virions. In this study, the C-terminal knob domain of the FAdV-4 Fiber2 protein was expressed and purified, and its trimer structure (PDB ID: 7W83) was determined for the first time. A series of affinity peptides targeting the knob domain of the Fiber2 protein were designed and synthesized on the basis of the crystal structure using computer virtual screening technology. A total of eight peptides were screened using an immunoperoxidase monolayer assay and RT-qPCR, and they exhibited strong binding affinities to the knob domain of the FAdV-4 Fiber2 protein in a surface plasmon resonance assay. Treatment with peptide number 15 (P15; WWHEKE) at different concentrations (10, 25, and 50 µM) significantly reduced the expression level of the Fiber2 protein and the viral titer during FAdV-4 infection. P15 was found to be an optimal peptide with antiviral activity against FAdV-4 in vitro with no cytotoxic effect on LMH cells up to 200 µM. This study led to the identification of a class of affinity peptides designed using computer virtual screening technology that targeted the knob domain of the FAdV-4 Fiber2 protein and may be developed as a novel potential and effective antiviral strategy in the prevention and control of FAdV-4.

Establishment of a lipid metabolism disorder model in ApoEb mutant zebrafish.

BACKGROUND AND AIMS: ApoEb is a zebrafish homologous to mammalian ApoE, whose deficiency would lead to lipid metabolism disorders (LMDs) like atherosclerosis. We attempted to knock out the zebrafish ApoEb, then establish a zebrafish model with LMD. METHODS: ApoEb was knocked out using the CRISPR/Cas9 system, and the accumulation of lipids was confirmed by Oil Red O staining, confocal imaging, and lipid measurements. The lipid-lowering effects of simvastatin (SIM), ezetimibe (EZE) and Xuezhikang (XZK), an extract derived from red yeast rice, were evaluated through in vivo imaging in zebrafish larvae. RESULTS: In the ApoEb mutant, significant vascular lipid deposition occurred, and lipid measurement performed in the whole-body homogenate of larvae and adult plasma showed significantly increased lipid levels. SIM, EZE and XZK apparently relieved hyperlipidemia in ApoEb mutants, and XZK had a significant inhibitory effect on the recruitment of neutrophils and macrophages. CONCLUSIONS: In this study, an LMD model has been established in ApoEb mutant zebrafish. We suggest that this versatile model could be applied in studying hypercholesterolemia and related vascular pathology in the context of early atherosclerosis, as well as the physiological function of ApoE.

Zfp36l1b protects angiogenesis through Notch1b/Dll4 and Vegfa regulation in zebrafish.

BACKGROUND AND AIMS: Angiogenesis is a key process for establishing functional vasculature during embryogenesis and involves different signaling mechanisms. The RNA binding protein Zfp36l1 was reported to be involved in various diseases in different species, including cardiovascular diseases. However, whether Zfp36l1b, one of the 2 paralogs of Zfp36l1 in zebrafish, works like mammalian Zfp36l1, and if the molecular mechanisms are different remains unclear. Here, we show that Zfp36l1b plays a crucial protective role in angiogenesis of zebrafish embryos. METHODS: We used transparent transgenic and wild-type zebrafish larvae to dynamically investigate the early stage of angiogenesis with confocal in vivo, after the knockdown of Zfp36l1b by morpholinos (MOs). In situ hybridization and fluorescence-activated cell sorting were performed to detect Zfp36l1b expression. mRNA rescue and CRISPR/Cas9 knockdown, and luciferase reporter experiments were performed to further explore the role of Zfp36l1b in angiogenesis. RESULTS: We found that knockdown of Zfp36l1b led to defected angiogenesis in intersomitic vessels and sub-intestinal veins (SIVs), which could be rescued by the addition of Zfp36l1b mRNA. Moreover, knockdown of Zfp36l1b suppressed Notch1b expression, while knockdown of Notch1b resulted in a partial relief of angiogenesis defects induced by Zfp36l1b down-regulation. Besides, Zfp36l1b knockdown alleviated the excessive branch of SIVs caused by Vegfa over-expression. CONCLUSIONS: Our results show that Zfp36l1b is responsible for establishing normal vessel circuits by affecting the extension of endothelial tip cells filopodia and the proliferation of endothelial cells partly through Notch1b/Fll4 suppression and synergistic function with Vegfa.

MicroRNA-126a Directs Lymphangiogenesis Through Interacting With Chemokine and Flt4 Signaling in Zebrafish.

OBJECTIVE: MicroRNA-126 (miR-126) is an endothelium-enriched miRNA and functions in vascular integrity and angiogenesis. The application of miRNA as potential biomarker and therapy target has been widely investigated in various pathological processes. However, its role in lymphatic diseases had not been widely explored. We aimed to reveal the role of miR-126 in lymphangiogenesis and the regulatory signaling pathways for potential targets of therapy. APPROACH AND RESULTS: Loss-of-function studies using morpholino oligonucleotides and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system showed that silencing of miR-126a severely affected the formation of parachordal lymphangioblasts and thoracic duct in zebrafish embryos, although their development in miR-126b knockdown embryos was normal. Expression analyses by in situ hybridization and immunofluorescence indicated that miR-126a was expressed in lymphatic vessels, as well as in blood vessels. Time-lapse confocal imaging assay further revealed that knockdown of miR-126a blocked both lymphangiogenic sprouts budding from the posterior cardinal vein and lymphangioblasts extension along horizontal myoseptum. Bioinformatics analysis and in vivo report assay identified that miR-126a upregulated Cxcl12a by targeting its 5' untranslated region. Moreover, loss- and gain-of-function studies revealed that Cxcl12a signaling acted downstream of miR-126a during parachordal lymphangioblast extension, whereby Flt4 signaling acts as a cooperator of miR-126a, allowing it to modulate lymphangiogenic sprout formation. CONCLUSIONS: These findings demonstrate that miR-126a directs lymphatic endothelial cell sprouting and extension by interacting with Cxcl12a-mediated chemokine signaling and Vegfc-Flt4 signal axis. Our results suggest that these key regulators of lymphangiogenesis may be involved in lymphatic pathogenesis of cardiovascular diseases.

Elevated microRNA-155 promotes foam cell formation by targeting HBP1 in atherogenesis.

AIM: MicroRNAs (miRNAs) play key roles in inflammatory responses of macrophages. However, the function of miRNAs in macrophage-derived foam cell formation is unclear. Here, we investigated the role of miRNAs in macrophage-derived foam cell formation and atherosclerotic development. METHODS AND RESULTS: Using quantitative reverse transcription-PCR (qRT-PCR), we found that the level of miR-155 expression was increased significantly in both plasma and macrophages from atherosclerosis (ApoE(-/-)) mice. We identified that oxidized low density lipoprotein (oxLDL) induced the expression and release of miR-155 in macrophages, and that miR-155 was required to mediate oxLDL-induced lipid uptake and reactive oxygen species (ROS) production of macrophages. Furthermore, ectopic overexpression and knockdown experiments identified that HMG box-transcription protein1 (HBP1) is a novel target of miR-155. Knockdown of HBP1 enhanced lipid uptake and ROS production in oxLDL-stimulated macrophages, and overexpression of HBP1 repressed these effects. Furthermore, bioinformatics analysis identified three YY1 binding sites in the promoter region of pri-miR-155 and verified YY1 binding directly to its promoter region. Detailed analysis showed that the YY1/HDAC2/4 complex negatively regulated the expression of miR-155 to suppress oxLDL-induced foam cell formation. Importantly, inhibition of miR-155 by a systemically delivered antagomiR-155 decreased clearly lipid-loading in macrophages and reduced atherosclerotic plaques in ApoE(-/-) mice. Moreover, we observed that the level of miR-155 expression was up-regulated in CD14(+) monocytes from patients with coronary heart disease. CONCLUSION: Our findings reveal a new regulatory pathway of YY1/HDACs/miR-155/HBP1 in macrophage-derived foam cell formation during early atherogenesis and suggest that miR-155 is a potential therapeutic target for atherosclerosis.