There is strong evidence from transgenic mouse studies that NRPs mediate angiogenesis. Mice overexpressing NRP1 were embryonic lethal and displayed several vascular abnormalities, such as excess capillaries and blood vessels, dilation of blood vessels, hemorrhage, and malformed hearts. The chimeric embryos appeared redder than their normal counterparts, suggesting that blood vessels were leaky, which was possibly caused by enhanced vascu lar permeability activity of VEGF165. It was concluded that expression of NRP1 was essential not only for neuronal development but also for development of the cardiovascular system.
The physiological role of NRPs in angiogenesis has also been determined using knockout mice. It was demonstrated that NRP1-deficient mutant mice were embryonic lethal between E12.5 to E13.5. In yolk sacs and embryos the vascular networks of large and small vessels were disorganized, the capillary networks were sparse, and normal branching did not occur. In the central nervous system (CNS), capillary invasion into the CNS was delayed and the capillary networks that were in the CNS were disorganized and had degenerated. The mutant embryos showed abnormal heart development, including lack of some of the branchial arch-related great vessels, and dorsal aorta and transposition of the aortic arch. The development of heart outflow tracts was also disturbed.
In another approach, the role of NRP1 in the vascular system was demonstrated by deleting NRP1 specifically from EC. Mutant mice were embryonic lethal by mid- to late gestation, with abnormal vasculature throughout the embryo. The larger vessels were intact, but medium and small vessels were missing. In the developing brain the vessels appeared larger and underdeveloped with very little branching, suggesting a defect in remodeling and branching of the primary vessel plexus. Taken together all of these data indicate that NRP1 is a critical receptor required for angio-genesis. On the other hand, NRP2 knockouts were viable into adulthood and did not display any abnormal vascular development.
More recently, double NRP1/NRP2 knockouts were reported. Transgenic mice, in which both NRP1 and NRP2 were targeted, died in utero at E8.5 and their yolk sacs were totally avascular. Mice that were homozygous for one gene but heterozygous for the other were also embryonic lethal and survived to E10 to E10.5. The vascular phenotypes of these mice were abnormal. The yolk sacs, while of normal size, displayed the absence of branching arteries and veins, the absence of a capillary bed, and the presence of large avascular spaces between the blood vessels. The embryos displayed blood vessels that were heterogenous in size, large avascular regions in the head and trunk, and unconnected blood vessel sprouts. The embryos were about 50 percent the length of wild-type mice and had multiple hemorrhages. These double NRP1/NRP2 knockout mice had a more severe abnormal vascular phenotype than either NRP1 or NRP2 single knockout mice. Their abnormal vascular phenotype resembled those of VEGF and VEGFR-2 knockouts. It appears that NRPs are early genes in embryonic vessel development, with overlapping functions that are required for normal blood vessel formation.
The zebrafish is an excellent system for analyzing vascular development. Zebrafish intersegmental vessels correspond to mammalian capillary sprouts, whereas the axial vessels correspond to larger blood vessels, such as arteries and veins (Figure 2A, control). The zebrafish NRP1 gene (znrpl) was isolated, and the zNRP1 protein was shown to be a functional receptor for human VEGF165. Whole-mount in situ hybridization showed that transcripts for znrpl during embryonic and early larval development were detected mainly in neuronal and vascular tissues. Knockdown of zNRP1 by using specific antisense oligos (Morpholino) in embryos resulted in severe defects in angiogenesis, including impaired circulation in the intersegmental vessels and dorsal longitudinal anastomotic vessel (DLAV) (Figure 2). However, circulation via the trunk artery and vein axial vessels, which are formed by vasculogenesis, was not affected. When zNRP1 and VEGF morpholinos were co-injected into embryos at concentrations that individually did not significantly inhibit blood vessel development, the result was a potent inhibition of blood cell circulation via both interseg-mental and axial vessels. These results demonstrated that VEGF and NRP1 act synergistically to promote a functional circulatory system. These results may provide a physiological demonstration that NRP1 regulates angiogenesis through a VEGF-dependent pathway.
Caudal Vein Trunk Axial Plexus
Arteries and Veins
Caudal Vein Trunk Axial Plexus
Arteries and Veins
B. NRP1 MO
Figure 2 NRP1 Knockdown in the Zebrafish: Microangiography. Fish embryos were injected with antisense morpholino (MO) or controls at the one- to four-cell stage. To visualize blood vessels flow, FITC-dextran was injected into the cardinal vein 56 hours postfertilization. (A) Normal circulation of the zebrafish. Injected with the four-base mismatch morpholino control. (B) In zebrafish injected with anti-zNRPl morpholino, blood flow via intersegmental vessels, DLAV, caudal vein plexus, and posterior vein is diminished. Axial vessel flow, however, is not affected.
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