Neurons secrete miR-132-containing exosomes to regulate brain vascular integrity.

Vascular integrity helps maintain brain microenvironment homeostasis, which is critical for the normal development and function of the central nervous system. It is known that neural cells can regulate brain vascular integrity. However, due to the high complexity of neurovascular interactions involved, understanding of the neural regulation of brain vascular integrity is still rudimentary. Using intact zebrafish larvae and cultured rodent brain cells, we find that neurons transfer miR-132, a highly conserved and neuron-enriched microRNA, via secreting exosomes to endothelial cells (ECs) to maintain brain vascular integrity. Following translocation to ECs through exosome internalization, miR-132 regulates the expression of vascular endothelial cadherin (VE-cadherin), an important adherens junction protein, by directly targeting eukaryotic elongation factor 2 kinase (eef2k). Disruption of neuronal miR-132 expression or exosome secretion, or overexpression of vascular eef2k impairs VE-cadherin expression and brain vascular integrity. Our study indicates that miR-132 acts as an intercellular signal mediating neural regulation of the brain vascular integrity and suggests that the neuronal exosome is a novel avenue for neurovascular communication.

Haemodynamics-driven developmental pruning of brain vasculature in zebrafish.

The brain blood vasculature consists of a highly ramified vessel network that is tailored to meet its physiological functions. How the brain vasculature is formed has long been fascinating biologists. Here we report that the developing vasculature in the zebrafish midbrain undergoes not only angiogenesis but also extensive vessel pruning, which is driven by changes in blood flow. This pruning process shapes the initial exuberant interconnected meshwork into a simplified architecture. Using in vivo long-term serial confocal imaging of the same zebrafish larvae during 1.5-7.5 d post-fertilization, we found that the early formed midbrain vasculature consisted of many vessel loops and higher order segments. Vessel pruning occurred preferentially at loop-forming segments via a process mainly involving lateral migration of endothelial cells (ECs) from pruned to unpruned segments rather than EC apoptosis, leading to gradual reduction in the vasculature complexity with development. Compared to unpruned ones, pruned segments exhibited a low and variable blood flow, which further decreased irreversibly prior to the onset of pruning. Local blockade of blood flow with micro-bead obstruction led to vessel pruning, whereas increasing blood flow by noradrenergic elevation of heartbeat impeded the pruning process. Furthermore, the occurrence of vessel pruning could be largely predicted by haemodynamics-based numerical simulation of vasculature refinement. Thus, changes of blood flow drive vessel pruning via lateral migration of ECs, leading to the simplification of the vasculature and possibly efficient routing of blood flow in the developing brain.

Celecoxib impairs heart development via inhibiting cyclooxygenase-2 activity in zebrafish embryos.

BACKGROUND: Celecoxib, a cyclooxygenase-2 inhibitor, is a commonly ingested drug that is used by some women during pregnancy. Although use of celecoxib is associated with increased cardiovascular risk in adults, its effect on fetal heart development remains unknown. METHODS: Zebrafish embryos were exposed to celecoxib or other relevant drugs from tailbud stage (10.3-72 h postfertilization). Heart looping and valve formation were examined at different developmental stages by in vivo confocal imaging. In addition, whole mount in situ hybridization was performed to examine drug-induced changes in the expression of heart valve marker genes. RESULTS: In celecoxib-treated zebrafish embryos, the heart failed to undergo normal looping and the heart valve was absent, causing serious blood regurgitation. Furthermore, celecoxib treatment disturbed the restricted expression of the heart valve markers bone morphogenetic protein 4 and versican-but not the cardiac chamber markers cardiac myosin light chain 2, ventricular myosin heavy chain, and atrial myosin heavy chain. These defects in heart development were markedly relieved by treatment with the cyclooxygenase-2 downstream product prostaglandin E2, and mimicked by the cyclooxygenase-2 inhibitor NS398, implying that celecoxib-induced heart defects were caused by the inhibition of cyclooxygenase-2 activity. CONCLUSIONS: These findings provide the first in vivo evidence that celecoxib exposure impairs heart development in zebrafish embryos by inhibiting cyclooxygenase-2 activity.

TRPC1 is essential for in vivo angiogenesis in zebrafish.

RATIONALE: Wiring vascular and neural networks are known to share common molecular signaling pathways. Activation of transient receptor potential type C channels (TRPCs) has recently been shown to underlie chemotropic guidance of neural axons. It is thus of interest to examine whether TRPCs are also involved in vascular development. OBJECTIVE: To determine the role of TRPC1 in angiogenesis in vivo during zebrafish development. METHODS AND RESULTS: Knockdown of zebrafish trpc1 by antisense morpholino oligonucleotides severely disrupted angiogenic sprouting of intersegmental vessels (ISVs) in zebrafish larvae. This angiogenic defect was prevented by overexpression of a morpholino oligonucleotide-resistant form of zebrafish trpc1 mRNA. Cell transplantation analysis showed that this requirement of Trpc1 for ISV growth was endothelial cell-autonomous. In vivo time-lapse imaging further revealed that the angiogenic defect was attributable to impairment of filopodia extension, migration, and proliferation of ISV tip cells. Furthermore, Trpc1 acted synergistically with vascular endothelial growth factor A (Vegf-a) in controlling ISV growth, and appeared to be downstream to Vegf-a in controlling angiogenesis, as evidence by the findings that Trpc1 was required for Vegf-a-induced ectopic angiogenesis of subintestinal veins and phosphorylation of extracellular signal-regulated kinase. CONCLUSIONS: These results provide the first in vivo evidence that TRPC1 is essential for angiogenesis, reminiscent of the role of TRPCs in axon guidance. It implicates that TRPC1 may represent a potential target for treating pathological angiogenesis.