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AIMS: MicroRNA-126 (miR-126), one of the most abundant microRNAs in platelets, is involved in the regulation of platelet activity and the circulating miR-126 is reduced during antiplatelet therapy. However, whether intraplatelet miR-126 plays a role in thrombosis and platelet inhibition remains unclear. METHODS AND RESULTS: Here, using tissue-specific knockout mice, we reported that the deficiency of miR-126 in platelets and vascular endothelial cells significantly prevented thrombosis and prolonged bleeding time. Using chimeric mice, we identified that the lack of intraplatelet miR-126 significantly prevented thrombosis. Ex vivo experiments further demonstrated that miR-126-deficient platelets displayed impaired platelet aggregation, spreading and secretory functions. Next, miR-126 was confirmed to target phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2) in platelet, which encodes a negative regulator of the PI3 K/AKT pathway, enhancing platelet activation through activating the integrin αIIbß3-mediated outside-in signaling. After undergoing myocardial infarction (MI), chimeric mice lacking intraplatelet miR-126 displayed reduced microvascular obstruction and prevented MI expansion in vivo. In contrast, overexpression of miR-126 by the administration of miR-126 agonist (agomiR-126) in wild-type mice aggravated microvascular obstruction and promoted MI expansion, which can be almost abolished by aspirin administration. In patients with cardiovascular diseases, antiplatelet therapies, either aspirin alone or combined with clopidogrel, decreased the level of intraplatelet miR-126. The reduction of intraplatelet miR-126 level was associated with the decrease of platelet activity. CONCLUSIONS: Our murine and human data reveal that (i) intraplatelet miR-126 contributes to platelet activity and promotes thrombus formation, and (ii) the reduction of intraplatelet miR-126 contributes to platelet inhibition during antiplatelet therapy.
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Di-(2-ethylhexyl) phthalate (DEHP) is among the most widely used plasticizers in plastic production, which has been detected in various environments. However, DEHP safety remains poorly known. Using zebrafish models, the effects of DEHP on the angiogenesis and hematopoiesis, and the underlying mechanism, were studied. Transgenic zebrafish embryos with specific fluorescence of vascular endothelial cells, myeloid cells, or hematopoietic stem cells were exposed to 0, 100, 150, 200, or 250 nM of DEHP for 22, 46 or 70 h, followed by fluorescence observation, endogenous alkaline phosphatase activity measurement, erythrocyte staining, and gene expression analysis by quantitative PCR and whole mount in situ hybridization. High DEHP concentrations decreased the sprouting rate, average diameter, and length, and the expansion area of the vessels lowered the EAP activity and suppressed the vascular endothelial growth factor (vegf) and hematopoietic marker genes, including c-myb, hbae1, hbbe1, and lyz expressions. DEHP treatment also decreased the number of hematopoietic stem cells, erythrocytes, and myeloid cells at 24 and 72 hpf. These DEHP-induced angiogenetic and hematopoietic defects might be alleviated by vegf overexpression. Our results reveal a plausible mechanistic link between DEHP exposure-induced embryonic angiogenetic defect and hematopoietic impairment.
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Dietilhexil Ftalato , Animales , Dietilhexil Ftalato/toxicidad , Pez Cebra , Factor A de Crecimiento Endotelial Vascular/genética , Células Endoteliales , Plastificantes , HematopoyesisRESUMEN
Myelopoiesis is the process in which the mature myeloid cells, including monocytes/macrophages and granulocytes, are developed. Irregular myelopoiesis may cause and deteriorate a variety of hematopoietic malignancies such as leukemia. Myeloid cells and their precursors are difficult to capture in circulation, let alone observe them in real time. For decades, researchers had to face these difficulties, particularly in in-vivo studies. As a unique animal model, zebrafish possesses numerous advantages like body transparency and convenient genetic manipulation, which is very suitable in myelopoiesis research. Here we review current knowledge on the origin and regulation of myeloid development and how zebrafish models were applied in these studies.
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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.
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Aterosclerosis , Hipercolesterolemia , Hiperlipidemias , Animales , Pez Cebra/metabolismo , Metabolismo de los Lípidos , Hipercolesterolemia/metabolismo , Ezetimiba , Aterosclerosis/patología , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Simvastatina/farmacología , Mamíferos/metabolismoRESUMEN
BACKGROUND: The compound Danshen Dripping Pill (CDDP), which is a mixture of extracts from Radix Salviae and Panax notoginseng, is a patented traditional Chinese medicine that is widely used in multiple countries for relieving coronary heart disease (CHD), but its pharmacological mechanism has not been fully elucidated. In this study, we screened the key pharmacological pathways and targets of CDDP that act on CHD using a network pharmacology-based strategy, and the angiogenic activity of CDDP was directly visually investigated in zebrafish embryos in vivo. METHODS: The potential therapeutic targets and pathways were predicted through a bioinformatics analysis. The proangiogenic effects of CDDP were examined using vascular sprouting assays on subintestinal vessels (SIVs) and optic arteries (OAs) as well as injury assays on intersegmental vessels (ISVs). Pharmacological experiments were applied to confirm the pathway involved. RESULTS: Sixty-five potential therapeutic targets of CDDP on CHD were identified and enriched in the PI3K/AKT and VEGF/VEGFR pathways. An in vivo study revealed that CDDP promoted angiogenesis in SIVs and OAs in a dose-dependent manner and relieved the impairments in ISVs induced by lenvatinib, a VEGF receptor kinase inhibitor (VRI). In addition, Vegfaa and Kdrl expression were significantly upregulated after CDDP treatment. Furthermore, the proangiogenic effect of CDDP could be abolished by PI3K/AKT pathway inhibitors. CONCLUSIONS: CDDP has a proangiogenic effect, the mechanism of which involves the VEGF/VEGFR and PI3K/AKT signaling pathways. These results suggest a new insight into the cardiovascular protective effect of CDDP.
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Fosfatidilinositol 3-Quinasas , Pez Cebra , Animales , Canfanos , Medicamentos Herbarios Chinos , Panax notoginseng , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/metabolismo , Salvia miltiorrhiza , Factor A de Crecimiento Endotelial Vascular/metabolismo , Pez Cebra/metabolismoRESUMEN
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.
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Células Endoteliales , Pez Cebra , Animales , Animales Modificados Genéticamente , Neovascularización Fisiológica , Proteínas de Pez Cebra/genéticaRESUMEN
Controlled by a strict mechanism, intracellular calcium (Ca(2+)) is closely related to various cellular activities, including the regulation of autophagy. Researchers believed that under normal or stress state, Ca(2+) has a positive or negative regulation effect on autophagy, the mechanisms of which are different. This bidirectional role of Ca(2+), promotive or suppressing in the regulation of autophagy under different conditions remains controversial, so as the potential mechanisms. Several studies reported that Ca(2+) promotes autophagy through plenty of ways, like inositol 1,4,5-trisphosphate receptor (IP3R) and beclin1 pathway, calmodulin-dependent kinase kinase beta (CaMKKß)-AMPK-mTOR pathway, mitochondrial energy metabolism-related Ca(2+) uptake, lysosome's regulation of Ca(2+) signal, and so on. Others thought Ca(2+) may inhibit autophagy through IP3R and beclin1-Bcl-2 complex and the AMPK-mTOR pathway, either. It seems to be still a long way to thoroughly understand the truth of Ca(2+) and autophagy.
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Autofagia , Calcio , Animales , Autofagia/fisiología , Calcio/química , Iones/química , Transducción de SeñalRESUMEN
Autophagy is crucial in the differentiation and development of both mammals and invertebrates, as a rapid response to environmental and hormonal stimuli. Autophagy is also important for intracellular renewal, maintaining the health of terminally differentiated cells. Studies of Drosophila, Caenorhabditis elegans, and other species revealed abnormal autophagy lead to developmental and differential abnormality, including those in salivary glands and midgut development, protein aggregation, removal of apoptotic cell corpses, and development of dauer and synapse. Autophagy also participates in the development of mammalian embryos before implantation into the uterus, adaption to the nascent hunger environment, blood cells production, and cell differentiation in adipogenesis. Autophagy found in various stem cells, like hematopoietic stem cells, bone marrow mesenchymal stem cells and neural stem cells (NSCs), is tightly associated with their self-renewal, directed differentiation, and senescence.