GPR124 regulates murine brain embryonic angiogenesis and BBB formation by an intracellular domain-independent mechanism.

The GPR124/RECK/WNT7 pathway is an essential regulator of CNS angiogenesis and blood-brain barrier (BBB) function. GPR124, a brain endothelial adhesion seven-pass transmembrane protein, associates with RECK, which binds and stabilizes newly synthesized WNT7 that is transferred to frizzled (FZD) to initiate canonical ß-catenin signaling. GPR124 remains enigmatic: although its extracellular domain (ECD) is essential, the poorly conserved intracellular domain (ICD) appears to be variably required in mammals versus zebrafish, potentially via adaptor protein bridging of GPR124 and FZD ICDs. GPR124 ICD deletion impairs zebrafish angiogenesis, but paradoxically retains WNT7 signaling upon mammalian transfection. We thus investigated GPR124 ICD function using the mouse deletion mutant Gpr124ΔC. Despite inefficiently expressed GPR124ΔC protein, Gpr124ΔC/ΔC mice could be born with normal cerebral cortex angiogenesis, in comparison with Gpr124-/- embryonic lethality, forebrain avascularity and hemorrhage. Gpr124ΔC/ΔC vascular phenotypes were restricted to sporadic ganglionic eminence angiogenic defects, attributable to impaired GPR124ΔC protein expression. Furthermore, Gpr124ΔC and the recombinant GPR124 ECD rescued WNT7 signaling in culture upon brain endothelial Gpr124 knockdown. Thus, in mice, GPR124-regulated CNS forebrain angiogenesis and BBB function are exerted by ICD-independent functionality, extending the signaling mechanisms used by adhesion seven-pass transmembrane receptors.

Retinoic acid signaling in mouse retina endothelial cells is required for early angiogenic growth.

The development of the retinal vasculature is essential to maintain health of the tissue, but the developmental mechanisms are not completely understood. The aim of this study was to investigate the cell-autonomous role of retinoic acid signaling in endothelial cells during retina vascular development. Using a temporal and cell-specific mouse model to disrupt retinoic acid signaling in endothelial cells in the postnatal retina (Pdgfbicre/+dnRAR403fl/fl mutants), we discovered that angiogenesis in the retina is significantly decreased with a reduction in retina vascularization, endothelial tip cell number and filipodia, and endothelial 'crowding' of stalk cells. Interestingly, by P15, the vasculature can overcome the early angiogenic defect and fully vascularized the retina. At P60, the vasculature is intact with no evidence of retina cell death or altered blood retinal barrier integrity. Further, we identified that the angiogenic defect seen in mutants at P6 correlates with decreased Vegfr3 expression in endothelial cells. Collectively, our work identified a previously unappreciated function for endothelial retinoic acid signaling in early retinal angiogenesis.

3D brain angiogenesis model to reconstitute functional human blood-brain barrier in vitro.

The human central nervous system (CNS) vasculature expresses a distinctive barrier phenotype, the blood-brain barrier (BBB). As the BBB contributes to low efficiency in CNS pharmacotherapy by restricting drug transport, the development of an in vitro human BBB model has been in demand. Here, we present a microfluidic model of CNS angiogenesis having three-dimensional (3D) lumenized vasculature in concert with perivascular cells. We confirmed the necessity of the angiogenic tri-culture system (brain endothelium in direct interaction with pericytes and astrocytes) to attain essential phenotypes of BBB vasculature, such as minimized vessel diameter and maximized junction expression. In addition, lower vascular permeability is achieved in the tri-culture condition compared to the monoculture condition. Notably, we focussed on reconstituting the functional efflux transporter system, including p-glycoprotein (p-gp), which is highly responsible for restrictive drug transport. By conducting the calcein-AM efflux assay on our 3D perfusable vasculature after treatment of efflux transporter inhibitors, we confirmed the higher efflux property and prominent effect of inhibitors in the tri-culture model. Taken together, we designed a 3D human BBB model with functional barrier properties based on a developmentally inspired CNS angiogenesis protocol. We expect the model to contribute to a deeper understanding of pathological CNS angiogenesis and the development of effective CNS medications.

Laminin-dystroglycan signaling regulates retinal arteriogenesis.

Proper arteriovenous morphogenesis is crucial for maintaining normal tissue perfusion. However, our understanding of how arterial morphogenesis is regulated in the CNS is incomplete. In this study, we asked whether vascular basement membrane (BM) laminins, specifically the γ3-containing isoforms, regulate retinal arterial morphogenesis. We provide evidence that Laminin-γ3 is deposited at both arterial and venous BMs during arteriogenesis. Vascular BM Laminin-γ3 bound dystroglycan (DG), a laminin receptor preferentially expressed by arterial endothelial cells (ECs) during arteriogenesis. Blockade of laminin-DG binding in vitro led to decreased Delta-like ligand (DLL)-4 expression in ECs. Moreover, genetic deletion of the Laminin-γ3- and EC-specific deletion of DG led to similar defects in retinal arteriogenesis, including reduced Dll4 expression, hyperbranching and reduced smooth muscle coverage. These results implicate a newly identified Laminin-γ3-DG signaling cascade that regulates arterial Dll4/Notch signaling to specify and stabilize retinal arteries.-Biswas, S., Watters, J., Bachay, G., Varshney, S., Hunter, D. D., Hu, H., Brunken, W. J. Laminin-dystroglycan signaling regulates retinal arteriogenesis.

BBB-on-a-Chip: Modeling Functional Human Blood-Brain Barrier by Mimicking 3D Brain Angiogenesis Using Microfluidic Chip.

Organ-on-a-chip enables human cell-based 3D tissue culture, which recapitulates the physiological structure and function of the tissue. In terms of the blood-brain barrier (BBB) modeling, the 3D structure of the vessel is essential for studying the cellular interactions among BBB composing cells and investigating the barrier function. Here, we describe a BBB-on-a-chip model with 3D perfusable human vasculature tri-cultured with pericytes and astrocytes. The culture method is based on mimicking angiogenic sprouting since the barrier formation is parallel with angiogenesis during the developmental process. This microfluidic-based 3D tri-culture system enables the comparative study on how surrounding BBB-related cells affect brain angiogenic sprouting. Moreover, the engineered perfusable vasculature is eligible for quantitative analysis on barrier function such as efflux transport system. We expect the BBB-on-a-chip could be used to enhance understanding BBB-related pathologies as well as the drug modulating barrier function of BBB.

Molecular determinants in Frizzled, Reck, and Wnt7a for ligand-specific signaling in neurovascular development.

The molecular basis of Wnt-Frizzled specificity is a central question in developmental biology. Reck, a multi-domain and multi-functional glycosylphosphatidylinositol-anchored protein, specifically enhances beta-catenin signaling by Wnt7a and Wnt7b in cooperation with the 7-transmembrane protein Gpr124. Among amino acids that distinguish Wnt7a and Wnt7b from other Wnts, two clusters are essential for signaling in a Reck- and Gpr124-dependent manner. Both clusters are far from the site of Frizzled binding: one resides at the amino terminus and the second resides in a protruding loop. Within Reck, the fourth of five tandem repeats of an unusual domain with six-cysteines (the CC domain) is essential for Wnt7a stimulation: substitutions P256A and W261A in CC4 eliminate this activity without changing protein abundance or surface localization. Mouse embryos carrying ReckP256A,W261A have severe defects in forebrain angiogenesis, providing the strongest evidence to date that Reck promotes CNS angiogenesis by specifically stimulating Wnt7a and Wnt7b signaling.

Reck and Gpr124 Are Essential Receptor Cofactors for Wnt7a/Wnt7b-Specific Signaling in Mammalian CNS Angiogenesis and Blood-Brain Barrier Regulation.

Reck, a GPI-anchored membrane protein, and Gpr124, an orphan GPCR, have been implicated in Wnt7a/Wnt7b signaling in the CNS vasculature. We show here that vascular endothelial cell (EC)-specific reduction in Reck impairs CNS angiogenesis and that EC-specific postnatal loss of Reck, combined with loss of Norrin, impairs blood-brain barrier (BBB) maintenance. The most N-terminal domain of Reck binds to the leucine-rich repeat (LRR) and immunoglobulin (Ig) domains of Gpr124, and weakening this interaction by targeted mutagenesis reduces Reck/Gpr124 stimulation of Wnt7a signaling in cell culture and impairs CNS angiogenesis. Finally, a soluble Gpr124(LRR-Ig) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Reck, and a soluble Reck(CC1-5) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Gpr124. These experiments indicate that Reck and Gpr124 are part of the cell surface protein complex that transduces Wnt7a- and Wnt7b-specific signals in mammalian CNS ECs to promote angiogenesis and regulate the BBB.