KIT is required for fetal liver erythropoiesis but dispensable for angiogenesis

Blood vessels are fundamental to sustain organ growth and tissue metabolism. In the mouse embryo, endothelial cell (EC) progenitors almost concomitantly give rise to the first blood vessels in the yolk sac and the large vessels of the embryo proper. Thereafter, the vascular network expands by angiogenesis to vascularize developing organs such as the brain. Although the first blood cells form in the yolk sac before blood vessels have assembled, consecutive waves of hematopoietic progenitors subsequently bud from hemogenic endothelium located within the wall of yolk sac and large intraembryonic vessels in a process termed endothelial to hematopoietic transition (endoHT). The receptor tyrosine kinase KIT is required for late embryonic erythropoiesis, but KIT is also expressed earlier in the hemogenic endothelium, in hematopoietic progenitors that arise via endoHT from hemogenic endothelium and non-hemogenic ECs, such as in the brain. However, it remains unclear whether KIT has essential roles in early hematopoiesis or even blood vessel growth. Here, we have combined transcriptomic analysis to delineate Kit expression with the analysis of knockout mice to show that KIT is expressed during but dispensable for yolk sac endoHT or brain angiogenesis but required for transient definitive erythropoiesis in the fetal liver.

H2S promotes developmental brain angiogenesis via the NOS/NO pathway in zebrafish

BackgroundHydrogen sulphide (H2S) is considered as the third member of the gasotransmitter family, along with nitric oxide (NO) and carbon monoxide. H2S has been reported to induce angiogenesis by promoting the growth, migration and tube-like structure formation of endothelial cells. Those studies were conducted in conditions of cell culture, mouse Matrigel plug assay model, rat wound healing model or rat hindlimb ischaemia model. Recent in vivo studies showed the physiological importance of H2S in muscle angiogenesis. However, the importance of endogenous H2S for brain angiogenesis during development remains unknown. We therefore aimed at determining the role of H2S in brain vascular development.Methods and resultsBoth knockdown and knockout of H2S-producing enzymes, cystathionine β-synthase (cbs) and cystathionine γ-lyase (cth), using morpholino oligonucleotides and clustered regularly interspaced short palindromic repeats/Cas9-mediated mutation, impaired brain vascular development of larval zebrafish. Incubation with the slow-releasing H2S donor GYY4137 alleviated the defects of brain vascular development in cbs and cth morphants. Quantitative analysis of the midbrain vascular network showed that H2S enhances angiogenesis without affecting the topological structure of the brain vasculature. Mechanically, nitric oxide synthase 2a (nos2a) expression and NO production were decreased in both cbs and cth morphants. Overexpression of nos2a by coinjection of cbs or cth MO with full-length zebrafish nos2a mRNA alleviated the brain vascular developmental defects in cbs and cth morphants.ConclusionWe conclude that H2S promotes brain developmental angiogenesis via the NOS/NO pathway in zebrafish.

Three-dimensional induced pluripotent stem-cell models of human brain angiogenesis

Background: During brain development, chemical cues released by developing neurons, cellular signaling with pericytes, and mechanical cues within the brain extracellular matrix (ECM) promote angiogenesis of brain microvascular endothelial cells (BMECs). Angiogenesis is also associated with diseases of the brain due to pathological chemical, cellular, and mechanical signaling. Existing in vitro and in vivo models of brain angiogenesis have key limitations. Methods: Here, we develop a high-throughput in vitro blood-brain barrier (BBB) bead assay of brain angiogenesis utilizing 150 μm diameter beads coated with induced pluripotent stem-cell (iPSC)-derived human BMECs (dhBMECs). After embedding the beads within a 3D matrix, we introduce various chemical cues and extracellular matrix components to explore their effects on angiogenic behavior. Based on the results from the bead assay, we generate a multi-scale model of the human cerebrovasculature within perfusable three-dimensional tissue-engineered blood-brain barrier microvessels.Results: A sprouting phenotype is optimized in confluent monolayers of dhBMECs using chemical treatment with vascular endothelial growth factor (VEGF) and wnt ligands, and the inclusion of pro-angiogenic ECM components. As a proof-of-principle that the bead angiogenesis assay can be applied to study pathological angiogenesis, we show that oxidative stress can exert concentration-dependent effects on angiogenesis. Finally, we demonstrate the formation of a hierarchical microvascular model of the human blood-brain barrier displaying key structural hallmarks. Conclusions: We develop two in vitro models of brain angiogenesis: the BBB bead assay and the tissue-engineered BBB microvessel model. These platforms provide a tool kit for studies of physiological and pathological brain angiogenesis, with key advantages over existing two-dimensional models.

Three-dimensional induced pluripotent stem-cell models of human brain angiogenesis

Background: During brain development, chemical cues released by developing neurons, cellular signaling with pericytes, and mechanical cues within the brain extracellular matrix (ECM) promotes angiogenesis occurs of brain microvascular endothelial cells (BMECs). During brain disease, angiogenesis can also occur due to pathological chemical, cellular, and mechanical signaling. Existing in vitro and in vivo models of brain angiogenesis have key limitations. Methods: Here, we develop a high-throughput in vitro BBB bead assay of brain angiogenesis utilizing 150 μm diameter beads coated with induced pluripotent stem-cell (iPSC)-derived human BMECs (dhBMECs). After embedding the beads within a 3D matrix, we introduce various chemical cues and extracellular matrix components to explore their effects on angiogenic behavior. Based on the results from the bead assay, we generate a multi-scale model of the human cerebrovasculature within perfusable three-dimensional tissue-engineered blood-brain barrier (BBB) microvessels. Results: A sprouting phenotype is optimized in confluent monolayers of dhBMECs using chemical treatment with vascular endothelial growth factor (VEGF) and wnt ligands, and the inclusion of pro-angiogenic ECM components. As a proof-of-principle that the bead angiogenesis assay can be applied to study pathological angiogenesis, we show that oxidative stress can exert concentration-dependent effects on angiogenesis. Finally, we demonstrate the formation of a hierarchical microvascular model of the human blood-brain barrier displaying key structural hallmarks. Conclusions: We develop two in vitro models of brain angiogenesis: the BBB bead assay and the tissue-engineered BBB microvessel model. These platforms provide a tool kit for studies of physiological and pathological brain angiogenesis, with key advantages over existing two-dimensional models.

Lab-On-A-Chip for the Development of Pro-/Anti-Angiogenic Nanomedicines to Treat Brain Diseases

There is a huge demand for pro-/anti-angiogenic nanomedicines to treat conditions such as ischemic strokes, brain tumors, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Nanomedicines are therapeutic particles in the size range of 10–1000 nm, where the drug is encapsulated into nano-capsules or adsorbed onto nano-scaffolds. They have good blood–brain barrier permeability, stability and shelf life, and able to rapidly target different sites in the brain. However, the relationship between the nanomedicines’ physical and chemical properties and its ability to travel across the brain remains incompletely understood. The main challenge is the lack of a reliable drug testing model for brain angiogenesis. Recently, microfluidic platforms (known as “lab-on-a-chip” or LOCs) have been developed to mimic the brain micro-vasculature related events, such as vasculogenesis, angiogenesis, inflammation, etc. The LOCs are able to closely replicate the dynamic conditions of the human brain and could be reliable platforms for drug screening applications. There are still many technical difficulties in establishing uniform and reproducible conditions, mainly due to the extreme complexity of the human brain. In this paper, we review the prospective of LOCs in the development of nanomedicines for brain angiogenesis–related conditions.