| Angiogenesis designates the formation of new vessels from preexisting ones,and occurs mainly during development.Tight control of this process is a prerequisite to avoid dysregulated vessel growth or remodeling(excess or defect in angiogenesis),the underlying causes of several diseases conditions.Thus,molecular insight into these patterning processes has significant biological and therapeutic implications.However,the molecular mechanisms that direct and control vascular growth processes are still incompletely understood.Growing lines of evidences have indicated that the vascular and the nervous systems of vertebrates share a high degree of anatomical similarity as well as the common guidance molecular system to ensure proper wiring.We are now aware that all four canonical families of neural guidance molecules and receptors,including Netrins and theirs receptors,have some members involved in angiogenesis.However,data reported led to the apparently contradictory conclusions for the roles of Netrin-1 as a pro-or an antiangiogenic factor.Recently we found for the first time that UNC5C,a Netrin-1 family receptor,is expressed in the developing vascular system,implying UNC5C might participate in vessel growth patterning.While few studies of UNC5C in vascular system were reported,function of UNC5C in angiogenesis remains to be elucidated due to the facts that no obvious defects in the vascular system have been observed and previous reports on its expression in cultured human endothelial cells(hECs)remains discrepancy.This study is to investigate the roles of UNC5C in the course of developmental angiogenesis.In our previous research,we detected the expression of Unc5c in chicken angioblast by utilizing semi quantitative RT-PCR assay.Moreover,we identified the UNC5C expression in three human endothelial cells through quantitative real-time PCR.We further demonstrated that UNC5C is selectively expressed in the endothelial cells of the developing vascular system in both chicken and zebrafish embryo by in situ hybridization.Interestingly,Unlike Netrin receptor UNC5B,the expression of UNC5C appeared largely restricted to the veins and capillaries,while weakly labelled in the arteries.Disruption of the Unc5c gene in chick embryo by the RCAS interference system,which is tailor-made for chickens with a modified chicken microRNA operon driven by a potent chicken U6 promoter,led to abnormal vessel branches and increased capillary sprouting both in yolk sac and in the embryo proper.We demonstrated also that the decrease of UNC5C promotes microvessel formation in an ex vivo chick aortic ring assay.Knocking down Unc5c in zebrafish by a splice blocking MO led to ectopic branching and path finding defects of intersegmental vessels,and this might not be associated with absence of the parachordal vessel that mentioned in previous reports.To further elucidate the function of UNC5C,we evaluated the effects of knockdown UNC5C on cultured human endothelial cells(HUVEC and HUAEC)in vitro.We found that knockdown of UNC5C stimulates tube formation,proliferation and migration of human endothelial cell in vitro.Since the signaling pathways downstream of UNC5C in ECs are not clear,we also made a preliminary study on the downstream signaling pathway of UNC5C,which may involve these proliferation,survival and migration effects.Our results indicated that knockdown of UNC5C receptor promotes angiogenesis through the activation of ERK and AKT signaling and suppression of Caspase-3 pathway.In conclusion,by loss of function and gain of function strategies in both in vivo and in vitro assays on chicken and zebrafish model,we preliminary identified UNC5C as a novel negative regulator in developmental angiogenesis,consistent with its role as a repulsive guidance molecular.Our research has gained new insight into the functions of Netrin/UNC5 ligand and receptor system,and offered some valuable information for cracking the confusion of Netrin-1/UNC5B. |
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