In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 110, No. 21 ( 2013-05-21)
Abstract:
The present study discloses a previously unknown effect of Nogo-A in the formation of blood vessels in the developing brain and retina. In particular, these findings demonstrate that Nogo-A plays an important role by discouraging the migration of vascular endothelial tip cells and the formation of blood vessels in the developing CNS. Thereby, these results shed light on the processes that regulate the density of the brain’s vasculature and expand the spectrum of molecules that influence the growth of nerve fibers and blood vessels by the CNS protein, Nogo-A. Because a variety of pathological processes that depend on angiogenesis ( 1 ) may be influenced by Nogo-A in the adult CNS, further studies should explore the possible role of Nogo-A in blood vessel repair after injury or stroke or in the vascularization of brain tumors. To analyze the functions of Nogo-A on the cellular level, we isolated vascular endothelial cells from enzymatically isolated microvessels of the postnatal mouse brain. When these cells were cultured on dishes coated with proteins extracted from brain, the adhesion and spreading of vascular endothelial cells were inhibited. This effect was attenuated when the Nogo-A protein, which was present in these extracts, was neutralized by antibodies or when the brain extracts were generated from mice in which Nogo-A was deleted genetically (Nogo-A KO). Purified Nogo-A inhibited vascular endothelial cell adhesion, spreading, migration, and vessel sprout formation in vitro. Furthermore, Nogo-A induced repulsive effects at the level of single endothelial cell filopodia, the actin-containing membrane protrusions that are important for in vivo pathfinding of vascular endothelial tip cells. The intracellular signaling molecules Ras homolog gene family, member A (Rho-A) and Rho-associated, coiled-coil containing protein kinase (ROCK), as well as Myosin II, played a role in mediating the repulsive effects of Nogo-A on the cellular cytoskeleton of endothelial cells. These results suggest that Nogo-A activates a Nogo-A–specific receptor on vascular endothelial cells that can inhibit endothelial cell motility in vitro and in vivo. In this study, we used a combination of in vivo experiments and in vitro assays to study the role of Nogo-A on angiogenesis and vascular endothelial cells in the developing mouse CNS. Nogo-A was found to be expressed in neurons in the postnatal brain and retina, in close proximity to vascular endothelial tip cells and their finger-like protrusions, filopodia ( Fig. P1 A ). At the postnatal stage, when angiogenesis is highly dynamic, CNS blood vessels grew in a tissue containing Nogo-A, but the vascular endothelial cells themselves did not show any Nogo-A expression ( Fig. P1 A ). Genetic deletion or antibody-mediated neutralization of Nogo-A in 4- or 8-d-old mice led to a significant increase in the number of endothelial tip cells in the tissue and to a higher tissue density of functional blood vessels in various brain regions as well as in the retina ( Fig. P1 B ). These results suggest that Nogo-A acts as a negative regulator of blood vessel growth in the CNS in vivo. The growing tips of nerve fibers and vascular endothelial tip cells are specialized structures that sense guidance and positional cues in the tissue microenvironment ( 2 ). Interestingly, several factors that regulate nerve fiber growth and guide specific axons also play similar roles for migrating vascular endothelial cells ( 1 , 2 ). Nogo-A is a CNS membrane protein that has been identified as a potent inhibitory molecule for injured adult neuron plasticity and axonal growth in vivo ( 3 ); for example, neutralizing Nogo-A with function-blocking antibodies enhances the regeneration of injured fiber tracts and the plastic rewiring of circuits and improves functional recovery in animal models of paraplegia ( 3 ) and stroke ( 4 ). A phase II clinical trial with anti–Nogo-A antibodies is currently in preparation for patients with spinal cord injuries ( 3 ). The physiological function of Nogo-A for nonneuronal cells in the CNS (e.g., for growing blood vessels) has been poorly investigated in vivo until now. We therefore explored whether Nogo-A influences the formation of blood vessels in the immature brain. Blood vessels and nerves are crucial for the proper function of every tissue and organ in the body. Interestingly, recent findings suggest that growing nerves and blood vessels are guided by similar mechanisms during development ( 1 , 2 ). The membrane protein Nogo-A, which is present mostly in the CNS, is an inhibitory factor that restricts axonal growth and regeneration after lesions in the adult CNS ( 3 ). Here, we show that Nogo-A also acts as a “brake” for growing blood vessels during their invasion of the developing brain or eye. These results reveal how the developing brain interacts with the most important supplier of its nutrients, the blood vessel system.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1216203110
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2013
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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