Erythro-myeloid progenitors contribute endothelial cells to blood vessels.

The earliest blood vessels in mammalian embryos are formed when endothelial cells differentiate from angioblasts and coalesce into tubular networks. Thereafter, the endothelium is thought to expand solely by proliferation of pre-existing endothelial cells. Here we show that a complementary source of endothelial cells is recruited into pre-existing vasculature after differentiation from the earliest precursors of erythrocytes, megakaryocytes and macrophages, the erythro-myeloid progenitors (EMPs) that are born in the yolk sac. A first wave of EMPs contributes endothelial cells to the yolk sac endothelium, and a second wave of EMPs colonizes the embryo and contributes endothelial cells to intraembryonic endothelium in multiple organs, where they persist into adulthood. By demonstrating that EMPs constitute a hitherto unrecognized source of endothelial cells, we reveal that embryonic blood vascular endothelium expands in a dual mechanism that involves both the proliferation of pre-existing endothelial cells and the incorporation of endothelial cells derived from haematopoietic precursors.

Targeting miR-27a/VE-cadherin interactions rescues cerebral cavernous malformations in mice.

Cerebral cavernous malformations (CCMs) are vascular lesions predominantly developing in the central nervous system (CNS), with no effective treatments other than surgery. Loss-of-function mutation in CCM1/krev interaction trapped 1 (KRIT1), CCM2, or CCM3/programmed cell death 10 (PDCD10) causes lesions that are characterized by abnormal vascular integrity. Vascular endothelial cadherin (VE-cadherin), a major regulator of endothelial cell (EC) junctional integrity is strongly disorganized in ECs lining the CCM lesions. We report here that microRNA-27a (miR-27a), a negative regulator of VE-cadherin, is elevated in ECs isolated from mouse brains developing early CCM lesions and in cultured ECs with CCM1 or CCM2 depletion. Furthermore, we show miR-27a acts downstream of kruppel-like factor (KLF)2 and KLF4, two known key transcription factors involved in CCM lesion development. Using CD5-2 (a target site blocker [TSB]) to prevent the miR-27a/VE-cadherin mRNA interaction, we present a potential therapy to increase VE-cadherin expression and thus rescue the abnormal vascular integrity. In CCM1- or CCM2-depleted ECs, CD5-2 reduces monolayer permeability, and in Ccm1 heterozygous mice, it restores dermal vessel barrier function. In a neonatal mouse model of CCM disease, CD5-2 normalizes vasculature and reduces vascular leakage in the lesions, inhibits the development of large lesions, and significantly reduces the size of established lesions in the hindbrain. Furthermore, CD5-2 limits the accumulation of inflammatory cells in the lesion area. Our work has established that VE-cadherin is a potential therapeutic target for normalization of the vasculature and highlights that targeting miR-27a/VE-cadherin interaction by CD5-2 is a potential novel therapy for the devastating disease, CCM.

Hindbrain neurovascular anatomy of adult goldfish (Carassius auratus).

The goldfish hindbrain develops from a segmented (rhombomeric) neuroepithelial scaffold, similar to other vertebrates. Motor, reticular and other neuronal groups develop in specific segmental locations within this rhombomeric framework. Teleosts are unique in possessing a segmental series of unpaired, midline central arteries that extend from the basilar artery and penetrate the pial midline of each hindbrain rhombomere (r). This study demonstrates that the rhombencephalic arterial supply of the brainstem forms in relation to the neural segments they supply. Midline central arteries penetrate the pial floor plate and branch within the neuroepithelium near the ventricular surface to form vascular trees that extend back towards the pial surface. This intramural branching pattern has not been described in any other vertebrate, with blood flow in a ventriculo-pial direction, vastly different than the pial-ventricular blood flow observed in most other vertebrates. Each central arterial stem penetrates the pial midline and ascends through the floor plate, giving off short transverse paramedian branches that extend a short distance into the adjoining basal plate to supply ventromedial areas of the brainstem, including direct supply of reticulospinal neurons. Robust r3 and r8 central arteries are significantly larger and form a more interconnected network than any of the remaining hindbrain vascular stems. The r3 arterial stem has extensive vascular branching, including specific vessels that supply the cerebellum, trigeminal motor nucleus located in r2/3 and facial motoneurons found in r6/7. Results suggest that some blood vessels may be predetermined to supply specific neuronal populations, even traveling outside of their original neurovascular territories in order to supply migrated neurons.

Regulation of embryonic neurogenesis by germinal zone vasculature.

In the adult rodent brain, new neurons are born in two germinal regions that are associated with blood vessels, and blood vessels and vessel-derived factors are thought to regulate the activity of adult neural stem cells. Recently, it has been proposed that a vascular niche also regulates prenatal neurogenesis. Here we identify the mouse embryo hindbrain as a powerful model to study embryonic neurogenesis and define the relationship between neural progenitor cell (NPC) behavior and vessel growth. Using this model, we show that a subventricular vascular plexus (SVP) extends through a hindbrain germinal zone populated by NPCs whose peak mitotic activity follows a surge in SVP growth. Hindbrains genetically defective in SVP formation owing to constitutive NRP1 loss showed a premature decline in both NPC activity and hindbrain growth downstream of precocious cell cycle exit, premature neuronal differentiation, and abnormal mitosis patterns. Defective regulation of NPC activity was not observed in mice lacking NRP1 expression by NPCs, but instead in mice lacking NRP1 selectively in endothelial cells, yet was independent of vascular roles in hindbrain oxygenation. Therefore, germinal zone vascularization sustains NPC proliferation in the prenatal brain.

Loss of Gspt1l disturbs the patterning of the brain central arteries in zebrafish.

The cranial vasculature is crucial for the survival and development of the central nervous system and is closely related to brain pathologies. Characterizations of the underlying mechanisms by which cranial vessels acquire their stereotypic patterning remain to be the key interest in the cerebrovascular research. In this report, we show an interesting zebrafish cq37 mutant displaying aberrant patterning of the central arteries. Genetic mapping results indicate that the gene responsible for cq37 encodes G1 to S phase transition 1, like (Gspt1l) with a nonsense mutation. Complementation studies with a CRISPR-generated allele, as well as mRNA rescues, together strongly demonstrate that gspt1l is the cq37 gene. Zebrafish gspt1l is broadly expressed in the brain with enhanced expression in hindbrain during central artery sprouting. Further studies reveal that vascular endothelial growth factor (VEGF) signaling and unfolded protein response (UPR) pathway are activated in gspt1lcq37 mutants. In addition, expression analysis shows that vegfa and activating transcription factor-4 (atf4) are strongly upregulated in regions of gspt1l expression. Our results suggest that loss of Gspt1l activates the UPR pathway, which in turn induces ectopic expression of vegfa via Atf4, thus disturbing the patterning of the central arteries.

Assembly and patterning of the vascular network of the vertebrate hindbrain.

The cranial vasculature is essential for the survival and development of the central nervous system and is important in stroke and other brain pathologies. Cranial vessels form in a reproducible and evolutionarily conserved manner, but the process by which these vessels assemble and acquire their stereotypic patterning remains unclear. Here, we examine the stepwise assembly and patterning of the vascular network of the zebrafish hindbrain. The major artery supplying the hindbrain, the basilar artery, runs along the ventral keel of the hindbrain in all vertebrates. We show that this artery forms by a novel process of medial sprouting and migration of endothelial cells from a bilateral pair of primitive veins, the primordial hindbrain channels. Subsequently, a second wave of dorsal sprouting from the primordial hindbrain channels gives rise to angiogenic central arteries that penetrate into and innervate the hindbrain. The chemokine receptor cxcr4a is expressed in migrating endothelial cells of the primordial hindbrain channels, whereas its ligand cxcl12b is expressed in the hindbrain neural keel immediately adjacent to the assembling basilar artery. Knockdown of either cxcl12b or cxcr4a results in defects in basilar artery formation, showing that the assembly and patterning of this crucial artery depends on chemokine signaling.

Neurovascular development and links to disease.

The developing central nervous system (CNS) is vascularized via ingression of blood vessels from the outside as the neural tissue expands. This angiogenic process occurs without perturbing CNS architecture due to exquisite cross-talk between the neural compartment and invading blood vessels. Subsequently, this intimate relationship also promotes the formation of the neurovascular unit that underlies the blood-brain barrier and regulates blood flow to match brain activity. This review provides a historical perspective on research into CNS blood vessel growth and patterning, discusses current models used to study CNS angiogenesis, and provides an overview of the cellular and molecular mechanisms that promote blood vessel growth and maturation. Finally, we highlight the significance of these mechanisms for two different types of neurovascular CNS disease.

Neurovascular development in the embryonic zebrafish hindbrain.

The brain is made of billions of highly metabolically active neurons whose activities provide the seat for cognitive, affective, sensory and motor functions. The cerebral vasculature meets the brain's unusually high demand for oxygen and glucose by providing it with the largest blood supply of any organ. Accordingly, disorders of the cerebral vasculature, such as congenital vascular malformations, stroke and tumors, compromise neuronal function and survival and often have crippling or fatal consequences. Yet, the assembly of the cerebral vasculature is a process that remains poorly understood. Here we exploit the physical and optical accessibility of the zebrafish embryo to characterize cerebral vascular development within the embryonic hindbrain. We find that this process is primarily driven by endothelial cell migration and follows a two-step sequence. First, perineural vessels with stereotypical anatomies are formed along the ventro-lateral surface of the neuroectoderm. Second, angiogenic sprouts derived from a subset of perineural vessels migrate into the hindbrain to form the intraneural vasculature. We find that these angiogenic sprouts reproducibly penetrate into the hindbrain via the rhombomere centers, where differentiated neurons reside, and that specific rhombomeres are invariably vascularized first. While the anatomy of intraneural vessels is variable from animal to animal, some aspects of the connectivity of perineural and intraneural vessels occur reproducibly within particular hindbrain locales. Using a chemical inhibitor of VEGF signaling we determine stage-specific requirements for this pathway in the formation of the hindbrain vasculature. Finally, we show that a subset of hindbrain vessels is aligned and/or in very close proximity to stereotypical neuron clusters and axon tracts. Using endothelium-deficient cloche mutants we show that the endothelium is dispensable for the organization and maintenance of these stereotypical neuron clusters and axon tracts in the early hindbrain. However, the cerebellum's upper rhombic lip and the optic tectum are abnormal in clo. Overall, this study provides a detailed, multi-stage characterization of early zebrafish hindbrain neurovascular development with cellular resolution up to the third day of age. This work thus serves as a useful reference for the neurovascular characterization of mutants, morphants and drug-treated embryos.

Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction.

Blood vessel networks expand in a 2-step process that begins with vessel sprouting and is followed by vessel anastomosis. Vessel sprouting is induced by chemotactic gradients of the vascular endothelial growth factor (VEGF), which stimulates tip cell protrusion. Yet it is not known which factors promote the fusion of neighboring tip cells to add new circuits to the existing vessel network. By combining the analysis of mouse mutants defective in macrophage development or VEGF signaling with live imaging in zebrafish, we now show that macrophages promote tip cell fusion downstream of VEGF-mediated tip cell induction. Macrophages therefore play a hitherto unidentified and unexpected role as vascular fusion cells. Moreover, we show that there are striking molecular similarities between the pro-angiogenic tissue macrophages essential for vascular development and those that promote the angiogenic switch in cancer, including the expression of the cell-surface proteins TIE2 and NRP1. Our findings suggest that tissue macrophages are a target for antiangiogenic therapies, but that they could equally well be exploited to stimulate tissue vascularization in ischemic disease.

The Embryonic Mouse Hindbrain Model to Study Sprouting Angiogenesis In Vivo.

Blood vessel growth is a fundamental process for organ development and wound healing but is also associated with ischemic diseases and cancer. The growth of new blood vessels from preexisting vasculature, termed sprouting angiogenesis, is the predominant mode of blood vessel growth in central nervous system vascularization and pathological vessel growth. Accordingly, studying the molecular and cellular mechanisms of angiogenesis holds the promise to find novel therapeutic targets to stimulate new vessel formation in ischemic tissues or inhibit pathological vessel growth in disease. The embryonic mouse hindbrain provides an excellent model to study sprouting angiogenesis in vivo by histochemical or fluorescent wholemount immunolabeling, thus allowing high-resolution image capture of nascent vasculature and subsequent quantification of relevant angiogenic parameters. This chapter describes how to use the mouse embryonic hindbrain as a model to study physiological angiogenesis, including detailed protocols for hindbrain dissection, wholemount staining, and angiogenic parameters analysis.

The Embryonic Mouse Hindbrain and Postnatal Retina as In Vivo Models to Study Angiogenesis.

Angiogenesis, the growth of new blood vessels from pre-existing ones, is a fundamental process for organ development, exercise-induced muscle growth, and wound healing, but is also associated with different diseases such as cancer and neovascular eye disease. Accordingly, elucidating the molecular and cellular mechanisms of angiogenesis has the potential to identify new therapeutic targets to stimulate new vessel formation in ischemic tissues or inhibit pathological vessel growth in disease. This chapter describes the mouse embryo hindbrain and postnatal retina as models to study physiological angiogenesis and provides detailed protocols for tissue dissection, sample staining and analysis.

Systematization, description, and territory of arteries that promote vascularization of the midbrain and rhombencephalon in the ostrich (Struthio camelus).

Brain specimens from 30 ostriches were injected with red-dyed latex via the internal carotid arteries (Aa.). The ventral tectal mesencephalic artery (a.), invariably a medium-sized single vessel, was, on the right side, a collateral branch of the caudal branch of the carotid artery (53.4%), a direct branch of the carotid artery (43.3%) and a direct branch of the basilar artery (3.3%) and on the left side, a collateral branch of the caudal branch of the carotid artery (66.7%), a direct branch of the carotid artery (30%), and a direct branch of the basilar artery (3.3%). It vascularized only the ventral half of the optic lobe, with no involvement in cerebellar vascularization on the right (93.3%) and left (80%) sides, extending to the ventrorostral-most cerebellar lobules, which were vascularized on the right (6.7%) and left (20%) sides. The caudal ventral cerebellar arteries were a single vessel on the right (96.7%) and left (93.3%) sides. Its first branch was a common trunk: dorsal spinal-caudal cerebellar on the right (60%) and left (56.6%) sides. Its second branch was the caudal cerebellar artery on the right (76.7%) and left (86.7%) sides. Its third branch was the second component of the caudal cerebellar artery on the right (6.7%) and left (3.3%) sides. The midbrain was vascularized by dorsal and ventral tectal mesencephalic arteries. The cerebellum was vascularized by branches of the caudal ventral cerebellar artery and by the dorsal cerebellar artery.

Detecting Three-Dimensional Vascular Networks in the Mouse Embryonic Hindbrain.

Blood vessel formation is a fine-regulated process and interfering with blood vessel formation causes embryonic lethality as well as associated with many diseases in the adult, including inflammatory, ischemic, and cancer metastatic diseases. Brain contains abundant blood vessels and has some unique physiological functions, such as blood-brain barrier. Due to the thickness and opaque characters of the tissues, it is a challenge to visualize the three-dimensional structures of the brain blood vessels in the mouse. Therefore, establishing a protocol to display the three-dimensional structures in the brain is required for exploring the regulatory molecular mechanisms in brain blood vessel formation. In this manuscript, we introduced a whole-mount and a vibratome thick section of mouse embryonic hindbrain to display the three-dimensional structures of brain vascular system.

Injury of an aberrant vertebral artery during a routine corpectomy: a case report and literature review.

CASE REPORT: A case report of a 58-year-old man who sustained a laceration of his left vertebral artery during a routine corpectomy for cervical myelopathy is reported. OBJECTIVE: To report iatrogenic injury of a tortuous vertebral artery during anterior cervical spine surgery and discuss appropriate diagnosis and treatment options for this complication. SETTING: UMass Memorial Medical Center, Worcester, MA, USA. BACKGROUND DATA: Vertebral artery anomalies, although rare, are typically present with degenerative processes and great care must be taken to avoid damage during a corpectomy. Cross-sectional imaging coupled with intraoperative angiography is helpful for the urgent evaluation of the injury site and identification of the contralateral vertebral artery's status. METHODS: This is a single case of a patient sustaining a laceration of the left vertebral artery during surgery, which resulted in a lateral medullary stroke. RESULTS: After the left vertebral artery laceration, hemostasis was achieved. With the intent to better visualize and possibly embolize or stent the injury, an angiographic study was carried out. The angiogram revealed a laceration of the left vertebral artery within the vertebral foramina at vertebral body level C6, but intact distal flow. The patient underwent angiographic embolization and a subsequent magnetic resonance imaging (MRI) revealed a left lateral medullary stroke consistent with the lack of flow through the left vertebral artery from C6 to the basilar artery. CONCLUSION: If a tortuous vertebral artery is suspected, then meticulous review of preoperative cross-sectional imaging should be implemented along with angiographic examination. If anomalies are detected and the standard procedure cannot be safely carried out, then alterations, such as preoperative stent placement, need to be considered.

Management of anterior inferior cerebellar artery aneurysms: an illustrative case and review of literature.

Aneurysms of the anterior inferior cerebellar artery (AICA) are relatively rare among intracranial aneurysms. They can occur in 1 of 3 regions of the AICA: 1) craniocaudal (high or low riding), 2) mediolateral-premeatal (proximal), and 3) meatal-postmeatal (distal). The management strategies for treatment differ according to the location and configuration of the aneurysm. The existing body of neurosurgical literature contains articles published on aneurysms arising from the AICA near the basilar artery (BA), intracanalicular/meatal aneurysms, and distal AICA. Several therapeutic options exist, encompassing microsurgical and endovascular techniques. The authors describe a case of treatment involving a large BA-AICA aneurysm approached via exposure of the presigmoid dura using a retromastoid suboccipital craniectomy and partial petrosectomy. Treatment of these lesions requires detailed knowledge of the anatomy, and an anatomical overview of the AICA with its arterial loops and significant branches is presented, including a discussion of the internal auditory (labyrinthine) artery, recurrent perforating arteries, subarcuate artery, and cerebellosubarcuate artery. The authors discuss the various surgical approaches (retromastoid, far lateral, subtemporal, and transclival) with appropriate illustrations, citing the advantages and disadvantages in accessing these AICA lesions in relation to these approaches. The complications of these different surgical techniques and possible clinical effects of parent artery occlusion during AICA surgery are highlighted.

Secretogranin-II plays a critical role in zebrafish neurovascular modeling.

Secretoneurin (SN) is a neuropeptide derived from specific proteolytic processing of the precursor secretogranin II (SgII). In zebrafish and other teleosts, there are two paralogs named sgIIa and sgIIb. Our results showed that neurons expressing sgIIb were aligned with central arteries in the hindbrain, demonstrating a close neurovascular association. Both sgIIb-/- and sgIIa-/-/sgIIb-/- mutant embryos were defective in hindbrain central artery development due to impairment of migration and proliferation of central artery cells. Further study revealed that sgIIb is non-cell autonomous and required for central artery development. Hindbrain arterial and venous network identities were not affected in sgIIb-/- mutant embryos, and the mRNA levels of Notch and VEGF pathway-related genes were not altered. However, the activation of MAPK and PI3K/AKT pathways was inhibited in sgIIb-/- mutant embryos. Reactivation of MAPK or PI3K/AKT in endothelial cells could partially rescue the central artery developmental defects in the sgIIb mutants. This study provides the first in vivo evidence that sgIIb plays a critical role in neurovascular modeling of the hindbrain. Targeting the SgII system may, therefore, represent a new avenue for the treatment of vascular defects in the central nervous system.

Elevated Expression of miR302-367 in Endothelial Cells Inhibits Developmental Angiogenesis via CDC42/CCND1 Mediated Signaling Pathways.

Rationale: Angiogenesis is critical for embryonic development and microRNAs fine-tune this process, but the underlying mechanisms remain incompletely understood. Methods: Endothelial cell (EC) specific miR302-367 line was used as gain-of-function and anti-miRs as loss-of-function models to investigate the effects of miR302-367 on developmental angiogenesis with embryonic hindbrain vasculature as an in vivo model and fibrin gel beads and tube formation assay as in vitro models. Cell migration was evaluated by Boyden chamber and scratch wound healing assay and cell proliferation by cell count, MTT assay, Ki67 immunostaining and PI cell cycle analysis. RNA high-throughput sequencing identified miR-target genes confirmed by chromatin immunoprecipitation and 3'-UTR luciferase reporter assay, and finally target site blocker determined the pathway contributing significantly to the phenotype observed upon microRNA expression. Results: Elevated EC miR302-367 expression reduced developmental angiogenesis, whereas it was enhanced by inhibition of miR302-367, possibly due to the intrinsic inhibitory effects on EC migration and proliferation. We identified Cdc42 as a direct target gene and elevated EC miR302-367 decreased total and active Cdc42, and further inhibited F-actin formation via the WASP and Klf2/Grb2/Pak1/LIM-kinase/Cofilin pathways. MiR302-367-mediated-Klf2 regulation of Grb2 for fine-tuning Pak1 activation contributing to the inhibited F-actin formation, and then the attenuation of EC migration. Moreover, miR302-367 directly down-regulated EC Ccnd1 and impaired cell proliferation via the Rb/E2F pathway. Conclusion: miR302-367 regulation of endothelial Cdc42 and Ccnd1 signal pathways for EC migration and proliferation advances our understanding of developmental angiogenesis, and meanwhile provides a rationale for future interventions of pathological angiogenesis that shares many common features of physiological angiogenesis.

Two anatomical autopsy cases of direct communication between a persistent primitive trigeminal artery and an anterior inferior cerebellar artery.

The persistent primitive trigeminal artery (PPTA) is the most common persistent carotid-basilar anastomosis. However, morphological findings of the PPTA based on the anatomical autopsy are very scarce. To understand the reason why such a variant artery develops, it is essential to examine the detailed morphology of the PPTA and developmental process of this artery. Here, we present two anatomical autopsy instances of the PPTA (cases 1 and 2). In the first case (78-year-old female; right side), the anterior inferior cerebellar artery (AICA) arose from the internal carotid artery passing medial to the abducens nerve. This artery gave off a small branch communicating to the basilar artery, passed lateral to the trigeminal nerve root, and continued backward to the dorsal surface of the cerebellum. Thus, in this case, the AICA is considered to be branched from the PPTA. In the second case (75-year-old female, left side), the PPTA branched from the internal carotid artery, and passed lateral to the abducens nerve, giving off an artery connecting with the AICA. These communicating arteries between the basilar artery and the AICA, recognized in cases 1 and 2, are considered to be the persistence of the primitive lateral basilovertebral anastomosis during the early embryological period. We propose that the primitive lateral basilovertebral anastomosis forms the arterial network around the trigeminal nerve root, and the AICA develops through this anastomosis.