Angiogenesis is a critical step in the growth of pancreatic neuroendocrine tumors (PNETs) and may be a selective target for PNET therapy. However, PNETs are robustly resistant to current anti-angiogenic therapies that primarily target the VEGFR pathway. Thus, the mechanism of PNET angiogenesis urgently needs to be clarified. Dataset analysis was used to identify angiogenesis-related genes in PNETs. Immunohistochemistry was performed to determine the relationship among Neuropilin 2 (NRP2), VEGFR2 and CD31. Cell proliferation, wound-healing and tube formation assays were performed to clarify the function of NRP2 in angiogenesis. The mechanism involved in NRP2-induced angiogenesis was detected by constructing plasmids with mutant variants and performing Western blot, and immunofluorescence assays. A mouse model was used to evaluate the effect of the NRP2 antibody in vivo, and clinical data were collected from patient records to verify the association between NRP2 and patient prognosis. NRP2, a VEGFR2 co-receptor, was positively correlated with vascularity but not with VEGFR2 in PNET tissues. NRP2 promoted the migration of human umbilical vein endothelial cells (HUVECs) cultured in the presence of conditioned medium PNET cells via a VEGF/VEGFR2-independent pathway. Moreover, NRP2 induced F-actin polymerization by activating the actin-binding protein cofilin. Cofilin phosphatase slingshot-1 (SSH1) was highly expressed in NRP2-activating cofilin, and silencing SSH1 ameliorated NRP2-activated HUVEC migration and F-actin polymerization. Furthermore, blocking NRP2 in vivo suppressed PNET angiogenesis and tumor growth. Finally, elevated NRP2 expression was associated with poor prognosis in PNET patients. Vascular NRP2 promotes PNET angiogenesis by activating the SSH1/cofilin/actin axis. Our findings demonstrate that NRP2 is an important regulator of angiogenesis and a potential therapeutic target of anti-angiogenesis therapy for PNET.

中文翻译：

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血管 NRP2 通过激活 SSH1-cofilin 轴触发 PNET 血管生成

血管生成是胰腺神经内分泌肿瘤 (PNET) 生长的关键步骤，可能是 PNET 治疗的选择性靶标。然而，PNETs 对目前主要针对 VEGFR 通路的抗血管生成疗法具有很强的抵抗力。因此，迫切需要阐明 PNET 血管生成的机制。数据集分析用于鉴定 PNET 中的血管生成相关基因。进行免疫组织化学以确定 Neuropilin 2 (NRP2)、VEGFR2 和 CD31 之间的关系。进行细胞增殖、伤口愈合和管形成测定以阐明 NRP2 在血管生成中的功能。通过构建具有突变体的质粒并进行蛋白质印迹和免疫荧光测定来检测参与 NRP2 诱导的血管生成的机制。使用小鼠模型评估 NRP2 抗体在体内的作用，并从患者记录中收集临床数据以验证 NRP2 与患者预后之间的关联。NRP2 是一种 VEGFR2 共受体，与血管分布呈正相关，但与 PNET 组织中的 VEGFR2 无关。NRP2 通过不依赖 VEGF/VEGFR2 的途径促进在条件培养基 PNET 细胞存在下培养的人脐静脉内皮细胞 (HUVEC) 的迁移。此外，NRP2 通过激活肌动蛋白结合蛋白 cofilin 诱导 F-肌动蛋白聚合。Cofilin 磷酸酶 slingshot-1 (SSH1) 在 NRP2 激活的 cofilin 中高度表达，沉默 SSH1 可改善 NRP2 激活的 HUVEC 迁移和 F-肌动蛋白聚合。此外，在体内阻断 NRP2 可抑制 PNET 血管生成和肿瘤生长。最后，NRP2 表达升高与 PNET 患者的不良预后相关。血管 NRP2 通过激活 SSH1/cofilin/actin 轴促进 PNET 血管生成。我们的研究结果表明，NRP2 是血管生成的重要调节因子，也是 PNET 抗血管生成治疗的潜在治疗靶点。