The formation and guidance of specialized endothelial tip cells is essential for both developmental and pathological angiogenesis. Notch-1 signalling regulates the generation of tip cells, which respond to gradients of vascular endothelial growth factor (VEGF-A). The molecular cues and signalling pathways that control the guidance of tip cells are poorly understood. Bidirectional signalling by Eph receptors and ephrin ligands represents one of the most important guidance cues involved in axon path finding. Here we show that ephrin-B2 reverse signalling involving PDZ interactions regulates endothelial tip cell guidance to control angiogenic sprouting and branching in physiological and pathological angiogenesis. In vivo, ephrin-B2 PDZ-signalling-deficient mice (ephrin-B2DeltaV) exhibit a reduced number of tip cells with fewer filopodial extensions at the vascular front in the mouse retina. In pathological settings, impaired PDZ signalling decreases tumour vascularization and growth. Mechanistically, we show that ephrin-B2 controls VEGF receptor (VEGFR)-2 internalization and signalling. Importantly, internalization of VEGFR2 is necessary for activation and downstream signalling of the receptor and is required for VEGF-induced tip cell filopodial extension. Together, our results suggest that ephrin-B2 at the tip cell filopodia regulates the proper spatial activation of VEGFR2 endocytosis and signalling to direct filopodial extension. Blocking ephrin-B2 reverse signalling may be an attractive alternative or combinatorial anti-angiogenic therapy strategy to disrupt VEGFR2 function in tumour angiogenesis.

Summary Vertebrate hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region from “hemogenic” endothelium. Here we show that the pro-inflammatory cytokine Ifn-γ and its receptor Crfb17 positively regulate HSC development in zebrafish. This regulation does not appear to modulate the proliferation or survival of HSCs or endothelial cells, but rather the endothelial to HSC transition. Notch signaling and blood flow positively regulate the expression of ifng and crfb17 in the AGM. Notably, Ifn-γ overexpression partially rescues the HSC loss observed in the absence of blood flow or Notch signaling. Importantly, Ifn-γ signaling acts cell-autonomously to control the endothelial to HSC transition. Ifn-γ activates Stat3, an atypical transducer of Ifn-γ signaling, in the AGM, and Stat3 inhibition decreases HSC formation. Together, our findings uncover a developmental role for an inflammatory cytokine and place its action downstream of Notch signaling and blood flow to control Stat3 activation and HSC emergence.

This protocol details a culture technique for neonatal mouse retina that allows the assessment and quantification of acute responses of developing blood vessels to pharmacological manipulation. The technique has proven to be a useful tool for elucidating the molecular mechanisms that underlie the guidance of tip cells in the complex scenario of the angiogenic sprouting process. This culture setting allows the acute stimulation or inhibition of cellular functions of endothelial cells in their physiological environment ex vivo. Compared with other existing techniques, such as retinal injections in animals, the explant culture described here is an easily manageable and highly flexible alternative that allows pharmacological manipulations of the developing retina vessels. The technique involves swift extraction of retina from intact eye and retinal flat mounting on a hydrophilic membrane with minimum disturbance of the tissue. The responses of tip endothelial cell sprouting activity and filopodial extension to different angiogenic and angioinhibitory factors can be evaluated within only 4 h. The whole process for the retinal explant cultures and stimulation can be completed in 10 h.

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Aims Dysregulated immune response contributes to inefficiency of treatment strategies to control hypertension and reduce the risk of end-organ damage. Uncovering the immune pathways driving the transition from the onset of hypertensive stimulus to the manifestation of multi-organ dysfunction are much-needed insights for immune targeted therapy. Methods and Results To aid visualization of cellular events orchestrating multi-organ pathogenesis, we modeled hypertensive cardiovascular remodeling in zebrafish. Zebrafish larvae exposed to ion-poor environment exhibited rapid angiotensinogen upregulation, followed by manifestation of arterial hypertension and cardiac remodeling that recapitulates key characteristics of incipient Heart Failure with preserved Ejection Fraction. In the brain, time-lapse imaging revealed the occurrence of cerebrovascular regression through endothelial retraction and migration in response to the ion-poor treatment. This phenomenon is associated with macrophage/microglia-endothelial contacts and endothelial junctional retraction. Cytokine and transcriptomic profiling identified systemic upregulation of interferon-γ and interleukin 1β, and revealed altered macrophage/microglia transcriptional program characterized by suppression of innate immunity and vasculo/neuroprotective gene expression. Both zebrafish and a murine model of pressure overload-induced brain damage demonstrated that the brain pathology and macrophage/microglia phenotypic alteration are dependent on interferon-γ signaling. In zebrafish, interferon-γ receptor 1 mutation prevents cerebrovascular remodeling and dysregulation of macrophage/microglia transcriptomic profile. Supplementation of bone morphogenetic protein 5, identified from the transcriptomic approach as a downregulated gene in ion-poor-treated macrophages/microglia that is rescued by interferon-γ blockage, mitigated cerebral microvessel loss. In mice subjected to transverse aortic constriction-induced pressure overload, typically developing cerebrovascular injury, neuroinflammation and cognitive dysfunction, interferon-γ neutralization protected them from blood-brain-barrier disruption, cerebrovascular rarefaction, and cognitive decline. Conclusions These findings uncover cellular and molecular players of an immune pathway communicating hypertensive stimulus to structural and functional remodeling of the brain and identify anti-interferon-γ treatment as a promising intervention strategy capable of preventing pressure overload-induced damage of the cerebrovascular and nervous systems. Translational Perspective Hypertension is a major risk factor for damages of the vasculature, heart, and brain, and thereby a major healthcare burden. Inadequate cerebral blood supply due to altered cerebrovascular structure and vasoregulatory disruption upon hypertension render the brain highly susceptible to stroke and cognitive decline. We envision that the cellular and molecular mechanisms uncovered here linking immune dysregulation to cerebrovascular remodeling and functional impairment of the brain will inform future development of immunomodulatory therapeutic strategies for counteracting derangement of macrophage/microglia activation and their vasculo/neuroprotective function in response to systemic inflammation in hypertension.