IntroductionMicroRNAs (miRNAs) are highly conserved, single-stranded noncoding short RNA molecules (18-24 nucleotides) that regulate gene expression at the posttranscriptional level. miRNAs silence gene expression by inhibiting the translation of proteins from mRNAs or by promoting the degradation of mRNAs. After transcription of the primary miRNA transcripts from the genome, their maturation is mediated by the 2 RNase III endonucleases Dicer and Drosha. Then, mature miRNAs are incorporated into the RNA-induced silencing complex, 1 which mediates the binding of the miRNA to the 3Ј-untranslated region (3Ј-UTR) of the target mRNA leading either to translational repression or degradation of the target mRNA. 2 Because miRNAs control specific expression patterns of target genes, miRNAs represent attractive candidates to interfere with neovascularization.Increasing evidence indicates that miRNAs are important regulators of vascular development and angiogenesis. 3,4 In this context, first studies addressed the function of the miRNAprocessing enzymes Dicer and/or Drosha to explore the general role of miRNAs for angiogenesis. Depletion of Dicer in zebrafish or mice revealed an aberrant vessel growth, and silencing of Dicer in endothelial cells reduced in vitro angiogenesis. [5][6][7] To date, several miRs that regulate endothelial cell function and angiogenesis have been identified, 8 including the pro-angiogenic miRs miR-130a, 9 miR-210, 5,10,11 and miR-378. 12 In addition, miR-126 was shown to regulate vascular integrity and angiogenesis during development and in ischemia-induced angiogenesis. [13][14][15] In contrast, miR-221 and miR-222, 7,16 miR-15 and miR-16, 17,18 and members of the miR-17-92 cluster 19,20 inhibit angiogenesis.In our previous study, we found that the members of the miR-23ϳ27ϳ24 cluster, miR-27a and miR-27b, were highly expressed in endothelial cells. 6 In addition, miR-27b was downregulated after Dicer and Drosha silencing, and inhibition of miR-27b significantly reduced endothelial cell sprouting in vitro, 6 indicating that miR-27b exerts pro-angiogenic effects. Recently, Zhou et al demonstrated that the miR-23ϳ27ϳ24 cluster regulates angiogenesis. 21 In muscle stem cells, miR-27b down-regulates Pax3 expression during myogenic differentiation. 22 Moreover, miR-27 down-regulates Runx1 expression during granulocyte differentiation 23 and the nuclear receptor peroxisome proliferatoractivated receptor-␥ (PPAR-␥) in adipocytes. 24 The myocyte enhancer factor 2C (MEF2C) is another important target of miR-27b during heart development. 25 However, the specific functions and targets of miR-27 in endothelial cells are largely unexplored. As the family members miR-27a and miR-27b differ in only one nucleotide and share the same seed sequence, we investigated the specific role of both family members for the angiogenic activity of endothelial cells and determined the effects on neovascularization. Here we identified the angiogenesis inhibitor semaphorin 6A as a The online version of this article contains a ...

Key Words: Rac1 Ⅲ ELMO1 Ⅲ DOCK180 Ⅲ vascular morphogenesis Ⅲ zebrafish A ngiogenesis, the formation of blood vessels from preexisting ones, is a crucial process during embryonic development as well as in several pathological conditions such as during tumor growth and in ischemic diseases. 1 Angiogenesis is activated by angiogenic growth factors, such as vascular endothelial growth factor (VEGF), leading to the formation of endothelial sprouts. 1 During the last few years it has been shown that angiogenic sprouts consist of 3 types of functionally different endothelial cell types, namely the tip cells, the stalk cells and the phalanx cells. 2 The tip cells are migratory cells at the leading front of the forming angiogenic sprouts; stalk cells are primarily formed by cell proliferation, and phalanx cells represent a more quiescent endothelium. Endothelial cell proliferation and migration have been intensively studied, and several important major signaling cascades have been identified. Based on the recognition of novel molecules regulating these major pathways 3 it is becoming increasingly important to identify and functional characterize the regulators for blood vessel formation. The small GTPases RhoA, Rac1, and Cdc42 have been shown to regulate migration of endothelial cells during angiogenesis. 4 -8 Rac1 is an essential factor during embryonic development as its endothelial specific deletion leads to an early embryonic vascular lethal phenotype. 7 Rac1 is ubiquitously expressed in 20 hpf zebrafish embryos 9 at the start phase of intersomitic vessel formation. 10 Therefore, a specific temporal and spatial regulation of Rac1 activation in the vascular system implies the existence of additional regulators for Rac1. Several important questions regarding the regulation and function of Rac1 in the embryonic and adult vasculature remain unanswered. Little is known about (1) which intracellular proteins modulate the activation of Rac1; (2) the upstream regulators of Rac1 in vivo are; and (3) the functions of these regulators in the vasculature in vivo are.The ELMO1/DOCK180 (engulfment and cell motility 1/dedicator of cytokinesis 180; also known as DOCK1) complex is an unusual GEF for Rac1 regulating cell migration in Caenorhabditis elegans, Drosophila melanogaster, and glioblastomas. 11,12 ELMO1 contains a pleckstrin homology domain for interaction with DOCK180. 13 DOCK180 uses a conserved "Docker" or "CZH2" domain to mediate the nucleotide exchange on Rac1. 11 Binding of ELMO1 to DOCK180 induces a conformational change releasing the DOCK180 self inhibitory loop. Consequently, the activated ELMO1/DOCK180 complex stabilizes Rac1 in its nucleotide free transition state and regulates its localization. 11 has not yet been reported. The aim of this study was to analyze the function of the ELMO1/DOCK180 complex in vascular development and to identify the upstream regulators of the bipartite GEF ELMO1/ DOCK180 in the vascular system. We report a strong, temporally controlled vascular expression of elmo1 in zebrafis...

Increased methylglyoxal (MG) formation is associated with diabetes and its complications. In zebrafish, knockout of the main MG detoxifying system Glyoxalase 1, led to limited MG elevation but significantly elevated aldehyde dehydrogenases (ALDH) activity and aldh3a1 expression, suggesting the compensatory role of Aldh3a1 in diabetes. To evaluate the function of Aldh3a1 in glucose homeostasis and diabetes, aldh3a1 −/− zebrafish mutants were generated using CRISPR-Cas9. Vasculature and pancreas morphology were analysed by zebrafish transgenic reporter lines. Corresponding reactive carbonyl species (RCS), glucose, transcriptome and metabolomics screenings were performed and ALDH activity was measured for further verification. Aldh3a1 −/− zebrafish larvae displayed retinal vasodilatory alterations, impaired glucose homeostasis, which can be aggravated via pdx1 silencing induced hyperglycaemia. Unexpectedly, MG was not altered, but 4-hydroxynonenal (4-HNE), another prominent lipid peroxidation RCS exhibited high affinity with Aldh3a1, was increased in aldh3a1 mutants. 4-HNE was responsible for the retinal phenotype via pancreas disruption induced hyperglycaemia and can be rescued via l -Carnosine treatment. Furthermore, in type 2 diabetic patients, serum 4-HNE was increased and correlated with disease progression. Thus, our data suggest impaired 4-HNE detoxification and elevated 4-HNE concentration as biomarkers but also the possible inducers for diabetes, from genetic susceptibility to the pathological progression.

Progression from the initial vascular response upon hyperglycemia to a proliferative stage with neovacularizations is the hallmark of proliferative diabetic retinopathy. Here, we report on the novel diabetic pdx1−/− zebrafish mutant as a model for diabetic retinopathy that lacks the transcription factor pdx1 through CRISPR-Cas9–mediated gene knockout leading to disturbed pancreatic development and hyperglycemia. Larval pdx1−/− mutants prominently show vasodilation of blood vessels through increased vascular thickness in the hyaloid network as direct developmental precursor of the adult retinal vasculature in zebrafish. In adult pdx1−/− mutants, impaired glucose homeostasis induces increased hyperbranching and hypersprouting with new vessel formation in the retina and aggravation of the vascular alterations from the larval to the adult stage. Both vascular aspects respond to antiangiogenic and antihyperglycemic pharmacological interventions in the larval stage and are accompanied by alterations in the nitric oxide metabolism. Thus, the pdx1−/− mutant represents a novel model to study mechanisms of hyperglycemia-induced retinopathy wherein extensive proangiogenic alterations in blood vessel morphology and metabolic alterations underlie the vascular phenotype.

JUNB, a subunit of the AP-1 transcription factor complex, mediates gene regulation in response to a plethora of extracellular stimuli. Previously, JUNB was shown to act as a critical positive regulator of blood vessel development and homeostasis as well as a negative regulator of proliferation, inflammation and tumour growth. Here, we demonstrate that the oncogenic miR-182 is a novel JUNB target. Loss-of-function studies by morpholino-mediated knockdown and the CRISPR/Cas9 technology identify a novel function for both JUNB and its target miR-182 in lymphatic vascular development in zebrafish. Furthermore, we show that miR-182 attenuates foxo1 expression indicating that strictly balanced Foxo1 levels are required for proper lymphatic vascular development in zebrafish. In conclusion, our findings uncover with the Junb/miR-182/Foxo1 regulatory axis a novel key player in governing lymphatic vascular morphogenesis in zebrafish.

Increased acrolein (ACR), a toxic metabolite derived from energy consumption, is associated with diabetes and its complications. However, the molecular mechanisms are mostly unknown, and a suitable animal model with internal increased ACR does not exist for in vivo studying so far. Several enzyme systems are responsible for acrolein detoxification, such as Aldehyde Dehydrogenase (ALDH), Aldo-Keto Reductase (AKR), and Glutathione S-Transferase (GST). To evaluate the function of ACR in glucose homeostasis and diabetes, akr1a1a −/− zebrafish mutants are generated using CRISPR/Cas9 technology. Accumulated endogenous acrolein is confirmed in akr1a1a −/− larvae and livers of adults. Moreover, a series of experiments are performed regarding organic alterations, the glucose homeostasis, transcriptome, and metabolomics in Tg(fli1:EGFP) zebrafish. Akr1a1a −/− larvae display impaired glucose homeostasis and angiogenic retina hyaloid vasculature, which are caused by reduced acrolein detoxification ability and increased internal ACR concentration. The effects of acrolein on hyaloid vasculature can be reversed by acrolein-scavenger l-carnosine treatment. In adult akr1a1a −/− mutants, impaired glucose tolerance accompanied by angiogenic retina vessels and glomerular basement membrane thickening, consistent with an early pathological appearance in diabetic retinopathy and nephropathy, are observed. Thus, the data strongly suggest impaired ACR detoxification and elevated ACR concentration as biomarkers and inducers for diabetes and diabetic complications.

b1-Integrins are essential for angiogenesis. The mechanisms regulating integrin function in endothelial cells (EC) and their contribution to angiogenesis remain elusive. Brag2 is a guanine nucleotide exchange factor for the small Arf-GTPases Arf5 and Arf6. The role of Brag2 in EC and angiogenesis and the underlying molecular mechanisms remain unclear. siRNA-mediated Brag2-silencing reduced EC angiogenic sprouting and migration. Brag2-siRNA transfection differentially affected a5b1-and aVb3-integrin function: specifically, Brag2-silencing increased focal/fibrillar adhesions and adhesion on b1-integrin ligands (fibronectin and collagen), while reducing the adhesion on the aVb3-integrin ligand, vitronectin. Consistent with these results, Brag2-silencing enhanced surface expression of a5b1-integrin, while reducing surface expression of aVb3-integrin. Mechanistically, Brag2-mediated aVb3-integrin-recycling and b1-integrin endocytosis and specifically of the active/matrix-bound a5b1-integrin present in fibrillar/focal adhesions (FA), suggesting that Brag2 contributes to the disassembly of FA via b1-integrin endocytosis. Arf5 and Arf6 are promoting downstream of Brag2 angiogenic sprouting, b1-integrin endocytosis and the regulation of FA. In vivo silencing of the Brag2-orthologues in zebrafish embryos using morpholinos perturbed vascular development. Furthermore, in vivo intravitreal injection of plasmids containing Brag2-shRNA reduced pathological ischemia-induced retinal and choroidal neovascularization. These data reveal that Brag2 is essential for developmental and pathological angiogenesis by promoting EC sprouting through regulation of adhesion by mediating b1-integrin internalization and link for the first time the process of b1-integrin endocytosis with angiogenesis.

The pdx1-/- zebrafish mutant was recently established as a novel animal model of diabetic retinopathy. Here, we investigate whether knockout of pdx1 also leads to diabetic kidney disease (DKD). pdx1-/- larvae exhibit several signs of early DKD such as glomerular hypertrophy, impairments in the filtration barrier corresponding to microalbuminuria and glomerular basement membrane (GBM) thickening. Adult pdx1-/- mutants show progressive GBM thickening in comparison to the larval state. Heterozygous pdx1 knockout also leads to glomerular hypertrophy as initial establishment of DKD similar to the pdx1-/- larvae. RNA sequencing (RNA-seq) of adult pdx1+/- kidneys uncovered regulations in multiple expected diabetic pathways related to podocyte disruption and hinting at early vascular dysregulation without obvious morphological alterations. Metabolome analysis and pharmacological intervention experiments revealed the contribution of phosphatidylethanolamine (PtdE) in the early establishment of kidney damage. In conclusion, this study identified the pdx1 mutant as a novel model for the study of DKD showing signs of the early disease progression already in larval stage and several selective features of later DKD in adult mutants.