Targeting of both neuropilin-1 and neuropilin-2 genes severely impairs developmental yolk sac embryonic angiogenesis

Department of Internal Medicine and Therapeutics, Department of Nutrition and Physiological Chemistry, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2002; 99(6):3657-62. DOI: 10.1073/pnas.022017899
Source: PubMed


Neuropilins (NP1 and NP2) are vascular endothelial growth factor (VEGF) receptors that mediate developmental and tumor angiogenesis. Transgenic mice, in which both NP1 and NP2 were targeted (NP1(-/-)NP2(-/-)) died in utero at E8.5. Their yolk sacs were totally avascular. Mice deficient for NP2 but heterozygous for NP1 (NP1(+/-)NP2(-/-)) or deficient for NP1 but heterozygous for NP2 (NP1(-/-)NP2(+/-)) were also embryonic lethal and survived to E10-E10.5. The E10 yolk sacs and embryos were easier to analyze for vascular phenotype than the fragile poorly formed 8.5 embryos. The vascular phenotypes of these E10 mice were very abnormal. The yolk sacs, although of normal size, lacked the larger collecting vessels and had less dense capillary networks. PECAM staining of yolk sac endothelial cells showed 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 heterogeneous in size, large avascular regions in the head and trunk, and blood vessel sprouts that were unconnected. The embryos were about 50% the length of wild-type mice and had multiple hemorrhages. These double NP1/NP2 knockout mice had a more severe abnormal vascular phenotype than either NP1 or NP2 single knockouts. Their abnormal vascular phenotype resembled those of VEGF and VEGFR-2 knockouts. These results suggest that NRPs are early genes in embryonic vessel development and that both NP1 and NP2 are required.

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Available from: Takashi Kitsukawa, May 22, 2014
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    • "Subsequently, NRP-1 was found to be a co-receptor for vascular endothelial growth factor 165 (VEGF 165) and is expressed in endothelial cells (EC), where it is involved in the regulation of angiogenesis and endothelial cell migration (7–9). Overexpression of NRP-1 in a transgenic mouse model increased capillary and blood vessel formation and resulted in hemorrhage (10), whereas functional inactivation of NRP-1 in mice led to embryonic lethality with multiple vascular abnormalities, including of avascular regions, heterogeneous blood vessel size and abnormally formed dorsal aorta (11). These results indicated that NRP-1 was a key regulator of developmental angiogenesis. "
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    ABSTRACT: Neuropilin-1 (NRP-1) is a novel receptor of vascular endothelial growth factor 165 that promotes angiogenesis, tumor growth, tumor invasion and metastasis. However, its role in tumorigenesis and progression of human hepatocellular carcinoma (HCC) is unknown. In this study, lentivirus-mediated short hairpin RNA (shRNA) was used to silence NRP-1 in the HCCLM6 cell line to explore its role in regulating the growth of HCC. Recombinant NRP-1 shRNA lentivirus was prepared and transfected into HCCLM6 cells. Transfection efficiencies of the lentivirus were observed by flow cytometry. Protein and mRNA expression of NRP-1 were examined by western blot analysis and quantitative reverse transcription-polymerase chain reaction (RT-PCR), and the effect of the lentivirus on cell growth was determined using MTT assay. Different cell groups were inoculated into nude mice to establish cancer xenografts, and tumor growth was monitored. Protein expression of NRP-1 in tumor tissues was detected by western blot assay. Microvessel density (MVD) in tumor tissues was assessed by immunohistochemistry (IHC). Lentivirus-mediated shRNA efficiently reduced endogenous NRP-1 expression in HCCLM6 cells and significantly inhibited cell growth in vitro. In vivo, NRP-1 knockdown in tumor tissues resulted in decreased vasculature. NRP-1 promotes the growth of HCC in vitro and in vivo, and therefore may be considered as a novel therapeutic target for HCC.
    Experimental and therapeutic medicine 01/2013; 5(1):150-154. DOI:10.3892/etm.2012.803 · 1.27 Impact Factor
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    • "The double knock-out of both NRP1 and NRP2 is characterized by an even more severe phenotype which is embryonic lethal around day E8. This is comparable to the complete knock-out of VEGF [8]. "
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    ABSTRACT: Neuropilin 1 (NRP1) is expressed on several cell types including neurons and endothelial cells, where it functions as an important regulator in development and during angiogenesis. As a cell surface receptor, NRP1 is able to bind to members of the VEGF family of growth factors and to secreted class 3 semaphorins. Neuropilin 1 is also highly expressed in keratinocytes, but the function of NRP1 in epidermal physiology and pathology is still unclear. To elucidate the role of NRP1 in skin in vivo we generated an epidermis-specific neuropilin 1 knock out mouse model by using the Cre-LoxP-System. Mice were viable and fertile and did not display any obvious skin or hair defects. After challenge with UVB irradiation, we found that deletion of epidermal NRP1 leads to increased rates of apoptosis both in vitro and in vivo. NRP1-deficient primary keratinocytes cultured in vitro showed significantly higher rates of apoptosis 24 hours after UVB. Likewise, there is a significant increase of active caspase 3 positive cells in the epidermis of Keratin 14-Cre-NRP1 (-/-) mice 24 hours after UVB irradiation. By Western Blot analysis we could show that NRP1 influences the cytosolic levels of Bcl-2, a pro-survival member of the Bcl-2 family. After UVB irradiation the amounts of Bcl-2 decrease in both protein extracts from murine epidermis and in NRP1-deficient keratinocytes in vitro, whereas wild type cells retain their Bcl-2 levels. Likewise, levels of phospho-Erk and Rac1 were lower in NRP1-knock out keratinocytes, whereas levels of pro-apoptotic p53 were higher. NRP1 expression in keratinocytes is dispensable for normal skin development. Upon UVB challenge, NRP1 contributes to the prevention of keratinocyte apoptosis. This pro-survival function of NRP1 is accompanied by the maintenance of high levels of the antiapoptotic regulator Bcl-2 and by lower levels of pro-apoptotic p53.
    PLoS ONE 12/2012; 7(12):e50944. DOI:10.1371/journal.pone.0050944 · 3.23 Impact Factor
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    • "To investigate the relationship of NRP1 and NRP2 signalling in SNS patterning , we examined mutants carrying the NC - specific Nrp1 - null mutation on a Nrp2 - null background . This strategy circumvented the lethality of full Nrp1 / Nrp2 - null mutants at E10 . 5 that is caused by insufficient yolk sac vascularisation ( Takashima et al . , 2002 ) . Thus , E13 . 5 compound null mutants were recovered at the expected Mendelian ratio ( observed frequency 3 / 40 Nrp1 fl / À Wnt1 - Creþ ; Nrp2 À / À in 6 litters , compared to the predicted fre - quency of 1 : 16 , i . e . , 3 / 48 ) . As observed for compound Sema3a / Sema3f - null mutants , Nrp1 fl / À Wnt1 - Cre ; Nrp2 À / À comb"
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    ABSTRACT: The sympathetic nervous system (SNS) arises from neural crest (NC) cells during embryonic development and innervates the internal organs of vertebrates to modulate their stress response. NRP1 and NRP2 are receptors for guidance cues of the class 3 semaphorin (SEMA) family and are expressed in partially overlapping patterns in sympathetic NC cells and their progeny. By comparing the phenotypes of mice lacking NRP1 or its ligand SEMA3A with mice lacking NRP1 in the sympathetic versus vascular endothelial cell lineages, we demonstrate that SEMA3A signalling through NRP1 has multiple cell-autonomous roles in SNS development. These roles include neuronal cell body positioning, neuronal aggregation and axon guidance, first during sympathetic chain assembly and then to regulate the innervation of the heart and aorta. Loss of NRP2 or its ligand SEMA3F impaired sympathetic gangliogenesis more mildly than loss of SEMA3A/NRP1 signalling, but caused ectopic neurite extension along the embryonic aorta. The analysis of compound mutants lacking SEMA3A and SEMA3F or NRP1 and NRP2 in the SNS demonstrated that both signalling pathways cooperate to organise the SNS. We further show that abnormal sympathetic development in mice lacking NRP1 in the sympathetic lineage has functional consequences, as it causes sinus bradycardia, similar to mice lacking SEMA3A.
    Developmental Biology 07/2012; 369(2):277-85. DOI:10.1016/j.ydbio.2012.06.026 · 3.55 Impact Factor
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