Ocular Angiogenesis Group, Departments of Ophthalmology and Medical Biology, Amsterdam Cardiovascular Sciences, Cancer Center Amsterdam, Amsterdam UMC, University of Amsterdam, Meibergdreef 9, Amsterdam, The Netherlands.
Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia.
Mol Biol Rep. 2020 Apr;47(4):2561-2572. doi: 10.1007/s11033-020-05339-0. Epub 2020 Mar 4.
We have previously identified insulin-like growth factor 2 (IGF2) and insulin-like growth factor 1 receptor (IGF1R) as essential proteins for tip cell maintenance and sprouting angiogenesis. In this study, we aim to identify other IGF family members involved in endothelial sprouting angiogenesis.
Effects on sprouting were analyzed in human umbilical vein endothelial cells (HUVECs) using the spheroid-based sprouting model, and were quantified as mean number of sprouts per spheroid and average sprout length. RNA silencing technology was used to knockdown gene expression. Recombinant forms of the ligands (IGF1 and IGF2, insulin) and the IGF-binding proteins (IGFBP) 3 and 4 were used to induce excess effects. Effects on the tip cell phenotype were analyzed by measuring the fraction of CD34 tip cells using flow cytometry and immunohistochemistry in a 3D angiogenesis model. Experiments were performed in the presence and absence of serum.
Knockdown of IGF2 inhibited sprouting in HUVECs, in particular when cultured in the absence of serum, suggesting that components in serum influence the signaling of IGF2 in angiogenesis in vitro. We then determined the effects of IGFBP3 and IGFBP4, which are both present in serum, on IGF2-IGF1R signaling in sprouting angiogenesis in the absence of serum: knockdown of IGFBP3 significantly reduced sprouting angiogenesis, whereas knockdown of IGFBP4 resulted in increased sprouting angiogenesis in both flow cytometry analysis and immunohistochemical analysis of the 3D angiogenesis model. Other IGF family members except INSR did not affect IGF2-IGF1R signaling.
Serum components and IGF binding proteins regulate IGF2 effects on sprouting angiogenesis. Whereas IGFBP3 acts as co-factor for IGF2-IGF1R binding, IGFBP4 inhibits IGF2 signaling.
我们之前已经确定胰岛素样生长因子 2（IGF2）和胰岛素样生长因子 1 受体（IGF1R）是维持尖端细胞和发芽血管生成所必需的蛋白质。在这项研究中，我们旨在确定其他参与血管生成发芽的 IGF 家族成员。
使用基于球体的发芽模型分析对球体的发芽的影响，并用每个球体的平均发芽数和平均发芽长度来量化。使用 RNA 沉默技术敲低基因表达。使用配体（IGF1 和 IGF2、胰岛素）和 IGF 结合蛋白（IGFBP）3 和 4 的重组形式诱导过量效应。通过流式细胞术和免疫组织化学在 3D 血管生成模型中测量 CD34 尖端细胞的分数来分析尖端细胞表型的影响。在有或没有血清的情况下进行实验。
IGF2 的敲低抑制了 HUVECs 的发芽，特别是在没有血清培养的情况下，这表明血清中的成分影响 IGF2 在体外血管生成中的信号传导。然后，我们确定了 IGFBP3 和 IGFBP4 的影响，这两种蛋白都存在于血清中，对无血清条件下 IGF2-IGF1R 信号在发芽血管生成中的作用：IGFBP3 的敲低显著降低了发芽血管生成，而 IGFBP4 的敲低导致在 3D 血管生成模型的流式细胞术分析和免疫组织化学分析中，发芽血管生成增加。除了 INSR 之外，其他 IGF 家族成员对 IGF2-IGF1R 信号没有影响。
血清成分和 IGF 结合蛋白调节 IGF2 对发芽血管生成的影响。虽然 IGFBP3 作为 IGF2-IGF1R 结合的辅助因子起作用，但 IGFBP4 抑制 IGF2 信号。
