A defined clathrin-mediated trafficking pathway regulates sFLT1/VEGFR1 secretion from endothelial cells.

FLT1/VEGFR1 negatively regulates VEGF-A signaling and is required for proper vessel morphogenesis during vascular development and vessel homeostasis. Although a soluble isoform, sFLT1, is often mis-regulated in disease and aging, how sFLT1 is trafficked and secreted from endothelial cells is not well understood. Here we define requirements for constitutive sFLT1 trafficking and secretion in endothelial cells from the Golgi to the plasma membrane, and we show that sFLT1 secretion requires clathrin at or near the Golgi. Perturbations that affect sFLT1 trafficking blunted endothelial cell secretion and promoted intracellular mis-localization in cells and zebrafish embryos. siRNA-mediated depletion of specific trafficking components revealed requirements for RAB27A, VAMP3, and STX3 for post-Golgi vesicle trafficking and sFLT1 secretion, while STX6, ARF1, and AP1 were required at the Golgi. Live-imaging of temporally controlled sFLT1 release from the endoplasmic reticulum showed clathrin-dependent sFLT1 trafficking at the Golgi into secretory vesicles that then trafficked to the plasma membrane. Depletion of STX6 altered vessel sprouting in 3D, suggesting that endothelial cell sFLT1 secretion influences proper vessel sprouting. Thus, specific trafficking components provide a secretory path from the Golgi to the plasma membrane for sFLT1 in endothelial cells that utilizes a specialized clathrin-dependent intermediate, suggesting novel therapeutic targets.

Interdependence of Angiogenesis and Arteriogenesis in Development and Disease.

The structure of arterial networks is optimized to allow efficient flow delivery to metabolically active tissues. Optimization of flow delivery is a continuous process involving synchronization of the structure and function of the microcirculation with the upstream arterial network. Risk factors for ischemic cardiovascular diseases, such as diabetes mellitus and hyperlipidemia, adversely affect endothelial function, induce capillary regression, and disrupt the micro- to macrocirculation cross-talk. We provide evidence showing that this loss of synchronization reduces arterial collateral network recruitment upon arterial stenosis, and the long-term clinical outcome of current revascularization strategies in these patient cohorts. We describe mechanisms and signals contributing to synchronized growth of micro- and macrocirculation in development and upon ischemic challenges in the adult organism and identify potential therapeutic targets. We conclude that a long-term successful revascularization strategy should aim at both removing obstructions in the proximal part of the arterial tree and restoring "bottom-up" vascular communication.

DGK and DZHK position paper on genome editing: basic science applications and future perspective.

For a long time, gene editing had been a scientific concept, which was limited to a few applications. With recent developments, following the discovery of TALEN zinc-finger endonucleases and in particular the CRISPR/Cas system, gene editing has become a technique applicable in most laboratories. The current gain- and loss-of function models in basic science are revolutionary as they allow unbiased screens of unprecedented depth and complexity and rapid development of transgenic animals. Modifications of CRISPR/Cas have been developed to precisely interrogate epigenetic regulation or to visualize DNA complexes. Moreover, gene editing as a clinical treatment option is rapidly developing with first trials on the way. This article reviews the most recent progress in the field, covering expert opinions gathered during joint conferences on genome editing of the German Cardiac Society (DGK) and the German Center for Cardiovascular Research (DZHK). Particularly focusing on the translational aspect and the combination of cellular and animal applications, the authors aim to provide direction for the development of the field and the most frequent applications with their problems.

Relief of hypoxia by angiogenesis promotes neural stem cell differentiation by targeting glycolysis.

Blood vessels are part of the stem cell niche in the developing cerebral cortex, but their in vivo role in controlling the expansion and differentiation of neural stem cells (NSCs) in development has not been studied. Here, we report that relief of hypoxia in the developing cerebral cortex by ingrowth of blood vessels temporo-spatially coincided with NSC differentiation. Selective perturbation of brain angiogenesis in vessel-specific Gpr124 null embryos, which prevented the relief from hypoxia, increased NSC expansion at the expense of differentiation. Conversely, exposure to increased oxygen levels rescued NSC differentiation in Gpr124 null embryos and increased it further in WT embryos, suggesting that niche blood vessels regulate NSC differentiation at least in part by providing oxygen. Consistent herewith, hypoxia-inducible factor (HIF)-1α levels controlled the switch of NSC expansion to differentiation. Finally, we provide evidence that high glycolytic activity of NSCs is required to prevent their precocious differentiation in vivo Thus, blood vessel function is required for efficient NSC differentiation in the developing cerebral cortex by providing oxygen and possibly regulating NSC metabolism.

Circular RNAs are a large class of animal RNAs with regulatory potency.

Circular RNAs (circRNAs) in animals are an enigmatic class of RNA with unknown function. To explore circRNAs systematically, we sequenced and computationally analysed human, mouse and nematode RNA. We detected thousands of well-expressed, stable circRNAs, often showing tissue/developmental-stage-specific expression. Sequence analysis indicated important regulatory functions for circRNAs. We found that a human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), is densely bound by microRNA (miRNA) effector complexes and harbours 63 conserved binding sites for the ancient miRNA miR-7. Further analyses indicated that CDR1as functions to bind miR-7 in neuronal tissues. Human CDR1as expression in zebrafish impaired midbrain development, similar to knocking down miR-7, suggesting that CDR1as is a miRNA antagonist with a miRNA-binding capacity ten times higher than any other known transcript. Together, our data provide evidence that circRNAs form a large class of post-transcriptional regulators. Numerous circRNAs form by head-to-tail splicing of exons, suggesting previously unrecognized regulatory potential of coding sequences.

Dll4-Notch signaling determines the formation of native arterial collateral networks and arterial function in mouse ischemia models.

Arteriogenesis requires growth of pre-existing arteriolar collateral networks and determines clinical outcome in arterial occlusive diseases. Factors responsible for the development of arteriolar collateral networks are poorly understood. The Notch ligand Delta-like 4 (Dll4) promotes arterial differentiation and restricts vessel branching. We hypothesized that Dll4 may act as a genetic determinant of collateral arterial networks and functional recovery in stroke and hind limb ischemia models in mice. Genetic loss- and gain-of-function approaches in mice showed that Dll4-Notch signaling restricts pial collateral artery formation by modulating arterial branching morphogenesis during embryogenesis. Adult Dll4(+/-) mice showed increased pial collateral numbers, but stroke volume upon middle cerebral artery occlusion was not reduced compared with wild-type littermates. Likewise, Dll4(+/-) mice showed reduced blood flow conductance after femoral artery occlusion, and, despite markedly increased angiogenesis, tissue ischemia was more severe. In peripheral arteries, loss of Dll4 adversely affected excitation-contraction coupling in arterial smooth muscle in response to vasopressor agents and arterial vessel wall adaption in response to increases in blood flow, collectively contributing to reduced flow reserve. We conclude that Dll4-Notch signaling modulates native collateral formation by acting on vascular branching morphogenesis during embryogenesis. Dll4 furthermore affects tissue perfusion by acting on arterial function and structure. Loss of Dll4 stimulates collateral formation and angiogenesis, but in the context of ischemic diseases such beneficial effects are overruled by adverse functional changes, demonstrating that ischemic recovery is not solely determined by collateral number but rather by vessel functionality.

Neuron navigator 3a regulates liver organogenesis during zebrafish embryogenesis.

Endodermal organogenesis requires a precise orchestration of cell fate specification and cell movements, collectively coordinating organ size and shape. In Caenorhabditis elegans, uncoordinated-53 (unc-53) encodes a neural guidance molecule that directs axonal growth. One of the vertebrate homologs of unc-53 is neuron navigator 3 (Nav3). Here, we identified a novel vertebrate neuron navigator 3 isoform in zebrafish, nav3a, and we provide genetic evidence in loss- and gain-of-function experiments showing its functional role in endodermal organogenesis during zebrafish embryogenesis. In zebrafish embryos, nav3a expression was initiated at 22 hpf in the gut endoderm and at 40 hpf expanded to the newly formed liver bud. Endodermal nav3a expression was controlled by Wnt2bb signaling and was independent of FGF and BMP signaling. Morpholino-mediated knockdown of nav3a resulted in a significantly reduced liver size, and impaired development of pancreas and swim bladder. In vivo time-lapse imaging of liver development in nav3a morphants revealed a failure of hepatoblast movement out from the gut endoderm during the liver budding stage, with hepatoblasts being retained in the intestinal endoderm. In hepatocytes in vitro, nav3a acts as a positive modulator of actin assembly in lamellipodia and filipodia extensions, allowing cellular movement. Knockdown of nav3a in vitro impeded hepatocyte movement. Endodermal-specific overexpression of nav3a in vivo resulted in additional ectopic endodermal budding beyond the normal liver and pancreatic budding sites. We conclude that nav3a is required for directing endodermal organogenesis involving coordination of endodermal cell behavior.

Flt1 acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo.

Endothelial tip cells guide angiogenic sprouts by exploring the local environment for guidance cues such as vascular endothelial growth factor (VegfA). Here we present Flt1 (Vegf receptor 1) loss- and gain-of-function data in zebrafish showing that Flt1 regulates tip cell formation and arterial branching morphogenesis. Zebrafish embryos expressed soluble Flt1 (sFlt1) and membrane-bound Flt1 (mFlt1). In Tg(flt1(BAC):yfp) × Tg(kdrl:ras-cherry)(s916) embryos, flt1:yfp was expressed in tip, stalk and base cells of segmental artery sprouts and overlapped with kdrl:cherry expression in these domains. flt1 morphants showed increased tip cell numbers, enhanced angiogenic behavior and hyperbranching of segmental artery sprouts. The additional arterial branches developed into functional vessels carrying blood flow. In support of a functional role for the extracellular VEGF-binding domain of Flt1, overexpression of sflt1 or mflt1 rescued aberrant branching in flt1 morphants, and overexpression of sflt1 or mflt1 in controls resulted in short arterial sprouts with reduced numbers of filopodia. flt1 morphants showed reduced expression of Notch receptors and of the Notch downstream target efnb2a, and ectopic expression of flt4 in arteries, consistent with loss of Notch signaling. Conditional overexpression of the notch1a intracellular cleaved domain in flt1 morphants restored segmental artery patterning. The developing nervous system of the trunk contributed to the distribution of Flt1, and the loss of flt1 affected neurons. Thus, Flt1 acts in a Notch-dependent manner as a negative regulator of tip cell differentiation and branching. Flt1 distribution may be fine-tuned, involving interactions with the developing nervous system.

Parenchymal cues define Vegfa-driven venous angiogenesis by activating a sprouting competent venous endothelial subtype.

Formation of organo-typical vascular networks requires cross-talk between differentiating parenchymal cells and developing blood vessels. Here we identify a Vegfa driven venous sprouting process involving parenchymal to vein cross-talk regulating venous endothelial Vegfa signaling strength and subsequent formation of a specialized angiogenic cell, prefabricated with an intact lumen and pericyte coverage, termed L-Tip cell. L-Tip cell selection in the venous domain requires genetic interaction between vascular Aplnra and Kdrl in a subset of venous endothelial cells and exposure to parenchymal derived Vegfa and Apelin. Parenchymal Esm1 controls the spatial positioning of venous sprouting by fine-tuning local Vegfa availability. These findings may provide a conceptual framework for understanding how Vegfa generates organo-typical vascular networks based on the selection of competent endothelial cells, induced via spatio-temporal control of endothelial Kdrl signaling strength involving multiple parenchymal derived cues generated in a tissue dependent metabolic context.

Inhibition of Notch signaling induces extensive intussusceptive neo-angiogenesis by recruitment of mononuclear cells.

Notch is an intercellular signaling pathway related mainly to sprouting neo-angiogenesis. The objective of our study was to evaluate the angiogenic mechanisms involved in the vascular augmentation (sprouting/intussusception) after Notch inhibition within perfused vascular beds using the chick area vasculosa and MxCreNotch1(lox/lox) mice. In vivo monitoring combined with morphological investigations demonstrated that inhibition of Notch signaling within perfused vascular beds remarkably induced intussusceptive angiogenesis (IA) with resultant dense immature capillary plexuses. The latter were characterized by 40 % increase in vascular density, pericyte detachment, enhanced vessel permeability, as well as recruitment and extravasation of mononuclear cells into the incipient transluminal pillars (quintessence of IA). Combination of Notch inhibition with injection of bone marrow-derived mononuclear cells dramatically enhanced IA with 80 % increase in vascular density and pillar number augmentation by 420 %. Additionally, there was down-regulation of ephrinB2 mRNA levels consequent to Notch inhibition. Inhibition of ephrinB2 or EphB4 signaling induced some pericyte detachment and resulted in up-regulation of VEGFRs but with neither an angiogenic response nor recruitment of mononuclear cells. Notably, Tie-2 receptor was down-regulated, and the chemotactic factors SDF-1/CXCR4 were up-regulated only due to the Notch inhibition. Disruption of Notch signaling at the fronts of developing vessels generally results in massive sprouting. On the contrary, in the already existing vascular beds, down-regulation of Notch signaling triggered rapid augmentation of the vasculature predominantly by IA. Notch inhibition disturbed vessel stability and led to pericyte detachment followed by extravasation of mononuclear cells. The mononuclear cells contributed to formation of transluminal pillars with sustained IA resulting in a dense vascular plexus without concomitant vascular remodeling and maturation.

Pulsatile shear and Gja5 modulate arterial identity and remodeling events during flow-driven arteriogenesis.

In the developing chicken embryo yolk sac vasculature, the expression of arterial identity genes requires arterial hemodynamic conditions. We hypothesize that arterial flow must provide a unique signal that is relevant for supporting arterial identity gene expression and is absent in veins. We analyzed factors related to flow, pressure and oxygenation in the chicken embryo vitelline vasculature in vivo. The best discrimination between arteries and veins was obtained by calculating the maximal pulsatile increase in shear rate relative to the time-averaged shear rate in the same vessel: the relative pulse slope index (RPSI). RPSI was significantly higher in arteries than veins. Arterial endothelial cells exposed to pulsatile shear in vitro augmented arterial marker expression as compared with exposure to constant shear. The expression of Gja5 correlated with arterial flow patterns: the redistribution of arterial flow provoked by vitelline artery ligation resulted in flow-driven collateral arterial network formation and was associated with increased expression of Gja5. In situ hybridization in normal and ligation embryos confirmed that Gja5 expression is confined to arteries and regulated by flow. In mice, Gja5 (connexin 40) was also expressed in arteries. In the adult, increased flow drives arteriogenesis and the formation of collateral arterial networks in peripheral occlusive diseases. Genetic ablation of Gja5 function in mice resulted in reduced arteriogenesis in two occlusion models. We conclude that pulsatile shear patterns may be central for supporting arterial identity, and that arterial Gja5 expression plays a functional role in flow-driven arteriogenesis.

Arteriogenesis is modulated by bradykinin receptor signaling.

RATIONALE: Positive outward remodeling of pre-existing collateral arteries into functional conductance arteries, arteriogenesis, is a major endogenous rescue mechanism to prevent cardiovascular ischemia. Collateral arterial growth is accompanied by expression of kinin precursor. However, the role of kinin signaling via the kinin receptors (B1R and B2R) in arteriogenesis is unclear. OBJECTIVE: The purpose of this study was to elucidate the functional role and mechanism of bradykinin receptor signaling in arteriogenesis. METHODS AND RESULTS: Bradykinin receptors positively affected arteriogenesis, with the contribution of B1R being more pronounced than B2R. In mice, arteriogenesis upon femoral artery occlusion was significantly reduced in B1R mutant mice as evidenced by reduced microspheres and laser Doppler flow perfusion measurements. Transplantation of wild-type bone marrow cells into irradiated B1R mutant mice restored arteriogenesis, whereas bone marrow chimeric mice generated by reconstituting wild-type mice with B1R mutant bone marrow showed reduced arteriogenesis after femoral artery occlusion. In the rat brain 3-vessel occlusion arteriogenesis model, pharmacological blockade of B1R inhibited arteriogenesis and stimulation of B1R enhanced arteriogenesis. In the rat, femoral artery ligation combined with arterial venous shunt model resulted in flow-driven arteriogenesis, and treatment with B1R antagonist R715 decreased vascular remodeling and leukocyte invasion (monocytes) into the perivascular tissue. In monocyte migration assays, in vitro B1R agonists enhanced migration of monocytes. CONCLUSIONS: Kinin receptors act as positive modulators of arteriogenesis in mice and rats. B1R can be blocked or therapeutically stimulated by B1R antagonists or agonists, respectively, involving a contribution of peripheral immune cells (monocytes) linking hemodynamic conditions with inflammatory pathways.

Thymosin ß4 and MRTF-A mitigate vessel regression despite cardiovascular risk factors.

Since clinical revascularization techniques of coronary or peripheral artery disease (CAD/PAD) focus on macrovessels of the heart, the microcirculatory compartment largely goes unnoticed. However, cardiovascular risk factors not only drive large vessel atherosclerosis, but also microcirculatory rarefaction, an instance unmet by current therapeutic schemes. Angiogenic gene therapy has the potential to reverse capillary rarefaction, but only if the disease-causing inflammation and vessel-destabilization are addressed. This review summarizes the current knowledge with regard to capillary rarefaction due to cardiovascular risk factors. Moreover, the potential of Thymosin ß4 (Tß4) and its downstream signal, myocardin-related transcription factor-A (MRTF-A), to counteract capillary rarefaction are discussed.

VEGF (Vascular Endothelial Growth Factor) Inhibition and Hypertension: Does Microvascular Rarefaction Play a Role?

Drugs acting by inhibition of the angiogenic action of VEGF (vascular endothelial growth factor) have become major instruments in the treatment of cancer. The downside of their favorable effects in cancer treatment is their frequent cardiovascular side effects. The most consistent finding thus far on the cardiovascular side effects of VEGF inhibitors is the high incidence of hypertension. In this short review, we discuss the evidence that hypertension occurring during VEGF inhibitor treatment is caused by microvascular rarefaction. After a review of the role of VEGF in microvascular growth and differentiation, we present evidence from studies in experimental models of hypertension as well as clinical studies on the microvascular network changes during and after VEGF inhibitor treatment.

A defined clathrin-mediated trafficking pathway regulates sFLT1/VEGFR1 secretion from endothelial cells.

FLT1/VEGFR1 negatively regulates VEGF-A signaling and is required for proper vessel morphogenesis during vascular development and vessel homeostasis. Although a soluble isoform, sFLT1, is often mis-regulated in disease and aging, how sFLT1 is trafficked and secreted from endothelial cells is not well understood. Here we define requirements for constitutive sFLT1 trafficking and secretion in endothelial cells from the Golgi to the plasma membrane, and we show that sFLT1 secretion requires clathrin at or near the Golgi. Perturbations that affect sFLT1 trafficking blunted endothelial cell secretion and promoted intracellular mis-localization in cells and zebrafish embryos. siRNA-mediated depletion of specific trafficking components revealed requirements for RAB27A, VAMP3, and STX3 for post-Golgi vesicle trafficking and sFLT1 secretion, while STX6, ARF1, and AP1 were required at the Golgi. Depletion of STX6 altered vessel sprouting in a 3D angiogenesis model, indicating that endothelial cell sFLT1 secretion is important for proper vessel sprouting. Thus, specific trafficking components provide a secretory path from the Golgi to the plasma membrane for sFLT1 in endothelial cells that utilizes a specialized clathrin-dependent intermediate, suggesting novel therapeutic targets.

Fetal growth restriction results in remodeled and less efficient hearts in children.

BACKGROUND: Fetal growth restriction (FGR) affects 5% to 10% of newborns and is associated with increased cardiovascular mortality in adulthood. The most commonly accepted hypothesis is that fetal metabolic programming leads secondarily to diseases associated with cardiovascular disease, such as obesity, diabetes mellitus, and hypertension. Our main objective was to evaluate the alternative hypothesis that FGR induces primary cardiac changes that persist into childhood. METHODS AND RESULTS: Within a cohort of fetuses with growth restriction identified in fetal life and followed up into childhood, we randomly selected 80 subjects with FGR and compared them with 120 normally grown fetuses, matched for gender, birth date, and gestational age at birth. Cardiovascular assessment was performed in childhood (mean age of 5 years). Compared with control subjects, children with FGR had a different cardiac shape, with increased transversal diameters and more globular cardiac ventricles. Although left ejection fraction was similar among the study groups, stroke volume was reduced significantly, which was compensated for by an increased heart rate to maintain output in severe FGR. This was associated with subclinical longitudinal systolic dysfunction (decreased myocardial peak velocities) and diastolic changes (increased E/E' ratio and E deceleration time). Children with FGR also had higher blood pressure and increased intima-media thickness. For all parameters evaluated, there was a linear increase with the severity of growth restriction. CONCLUSIONS: These findings suggest that FGR induces primary cardiac and vascular changes that could explain the increased predisposition to cardiovascular disease in adult life. If these results are confirmed, the impact of strategies with beneficial effects on cardiac remodeling should be explored in children with FGR.

Micro-RNA 92a as a Therapeutic Target for Cardiac Microvascular Dysfunction in Diabetes.

Microvascular dysfunction is a pathological hallmark of diabetes, and is central to the ethology of diabetes-associated cardiac events. Herein, previous studies have highlighted the role of the vasoactive micro-RNA 92a (miR-92a) in small, as well as large, animal models. In this study, we explore the effects of miR-92a on mouse and human cardiac microvascular endothelial cells (MCMEC, HCMEC), and its underlying molecular mechanisms. Diabetic HCMEC displayed impaired angiogenesis and a pronounced inflammatory phenotype. Quantitative PCR (qPCR) showed an upregulation of miR-92a in primary diabetic HCMEC. Downregulation of miR-92a by antagomir transfection in diabetic HCMEC rescued angiogenesis and ameliorated diabetic endothelial bed inflammation. Furthermore, additional analysis of potential in silico-identified miR-92a targets in diabetic HCMEC revealed the miR-92a dependent downregulation of an essential metalloprotease, ADAM10. Accordingly, downregulation of ADAM10 impaired angiogenesis and wound healing in MCMEC. In myocardial tissue slices from diabetic pigs, ADAM10 dysregulation in micro- and macro-vasculature could be shown. Altogether, our data demonstrate the role of miR-92a in cardiac microvascular dysfunction and inflammation in diabetes. Moreover, we describe for the first time the metalloprotease ADAM10 as a novel miR-92a target, mediating its anti-angiogenic effect.

In Vivo Behavior of the Antibacterial Peptide Cyclo[RRRWFW], Explored Using a 3-Hydroxychromone-Derived Fluorescent Amino Acid.

Labeling biomolecules with fluorescent labels is an established tool for structural, biochemical, and biophysical studies; however, it remains underused for small peptides. In this work, an amino acid bearing a 3-hydroxychromone fluorophore, 2-amino-3-(2-(furan-2-yl)-3-hydroxy-4-oxo-4H-chromen-6-yl)propanoic acid (FHC), was incorporated in a known hexameric antimicrobial peptide, cyclo[RRRWFW] (cWFW), in place of aromatic residues. Circular dichroism spectropolarimetry and antibacterial activity measurements demonstrated that the FHC residue perturbs the peptide structure depending on labeling position but does not modify the activity of cWFW significantly. FHC thus can be considered an adequate label for studies of the parent peptide. Several analytical and imaging techniques were used to establish the activity of the obtained labeled cWFW analogues toward animal cells and to study the behavior of the peptides in a multicellular organism. The 3-hydroxychromone fluorophore can undergo excited-state intramolecular proton transfer (ESIPT), resulting in double-band emission from its two tautomeric forms. This feature allowed us to get insights into conformational equilibria of the labeled peptides, localize the cWFW analogues in human cells (HeLa and HEK293) and zebrafish embryos, and assess the polarity of the local environment around the label by confocal fluorescence microscopy. We found that the labeled peptides efficiently penetrated cancerous cells and localized mainly in lipid-containing and/or other nonpolar subcellular compartments. In the zebrafish embryo, the peptides remained in the bloodstream upon injection into the cardinal vein, presumably adhering to lipoproteins and/or microvesicles. They did not diffuse into any tissue to a significant extent during the first 3 h after administration. This study demonstrated the utility of fluorescent labeling by double-emission labels to evaluate biologically active peptides as potential drug candidates in vivo.