Department of Immunology and Transfusion Medicine, Akershus University Hospital, University of Oslo, Norway.
Department of Biosciences, University of Oslo, Norway.
Biomed Res Int. 2019 Sep 10;2019:5496197. doi: 10.1155/2019/5496197. eCollection 2019.
Vascular endothelial growth factor (VEGF) receptors (VEGFR1 and VEGFR2) bind VEGF-A with high affinity. This study sought to determine the relative contributions of these two receptors to receptor-mediated endocytosis of VEGF-A and to clarify their endocytic itineraries in rat liver sinusoidal endothelial cells (LSECs).
Isolated LSECs and radiolabeled VEGF-A were used to examine surface binding and receptor-mediated endocytosis. Quantitative real time RT-PCR (Q-RT-PCR) and Western blotting were applied to demonstrate receptor expression.
Q-RT-PCR analysis showed that VEGFR1 and VEGFR2 mRNA were expressed in LSECs. Ligand saturation analysis at 4°C indicated two different classes of [I]-VEGFA binding sites on LSECs with apparent dissociation constants of 8 and 210 pM. At 37°C, LSECs efficiently took up and degraded [I]-VEGF-A for at least 2 hours. Uptake of [I]-VEGF-A by LSECs was blocked by dynasore that inhibits dynamin-dependent internalization, whereas inhibition of cysteine proteases by leupeptin inhibited degradation without affecting the uptake of [I]-VEGF-A, suggesting that it is degraded following transport to lysosomes. Incubation of LSECs in the continued presence of a saturating concentration of unlabeled VEGF-A at 37°C was associated with a loss of as much as 75% of the total VEGFR2 within 30 min as shown by Western blot analysis, whereas there was no appreciable decrease in protein levels for VEGFR1 after 120 min incubation, suggesting that VEGF-A stimulation downregulates VEGFR2, but not VEGFR1, in LSECs. This possibility was supported by the observation that a hexapeptide that specifically blocks VEGF-A binding to VEGFR1 caused a marked reduction in the uptake of [I]-VEGF-A, whereas a control peptide had no effect. Finally, live cell imaging studies using a fluorescently labeled anti-VEGFR2 antibody showed that VEGFR2 was transported via early and late endosomes to reach endolysosomes where degradation of the VEGFR2 takes place.
Our studies suggest that, subsequent to VEGF-A binding and internalization, the unoccupied VEGFR1 may recycle to the cell surface allowing its reutilization, whereas the majority of the internalized VEGFR2 is targeted for degradation.
血管内皮生长因子（VEGF）受体（VEGFR1 和 VEGFR2）与 VEGF-A 具有高亲和力结合。本研究旨在确定这两种受体在 VEGF-A 的受体介导内吞作用中的相对贡献，并阐明它们在大鼠肝窦内皮细胞（LSEC）中的内吞途径。
使用分离的 LSEC 和放射性标记的 VEGF-A 来检测表面结合和受体介导的内吞作用。应用定量实时 RT-PCR（Q-RT-PCR）和 Western 印迹来证明受体表达。
Q-RT-PCR 分析表明 VEGFR1 和 VEGFR2 mRNA 在 LSEC 中表达。在 4°C 时的配体饱和分析表明 LSEC 上有两种不同类别的 [I]-VEGFA 结合位点，其表观解离常数分别为 8 和 210 pM。在 37°C 时，LSEC 能够有效摄取和降解 [I]-VEGF-A，至少 2 小时。dynasore 可阻断 LSEC 摄取 [I]-VEGF-A，dynasore 可抑制依赖胞吞作用的动力蛋白，而亮抑蛋白酶抑制半胱氨酸蛋白酶的活性不影响 [I]-VEGF-A 的摄取，但抑制了其降解，表明其在转运到溶酶体后被降解。在 37°C 时，在持续存在饱和浓度未标记 VEGF-A 的情况下孵育 LSEC 会导致多达 75%的 VEGFR2 在 30 分钟内丢失，如 Western 印迹分析所示，而在 120 分钟孵育后，VEGFR1 的蛋白水平没有明显下降，表明 VEGF-A 刺激下调 LSEC 中的 VEGFR2，但不下调 VEGFR1。这种可能性得到了观察结果的支持，即一种特异性阻断 VEGF-A 与 VEGFR1 结合的六肽会导致 [I]-VEGF-A 的摄取显著减少，而对照肽则没有影响。最后，使用荧光标记的抗 VEGFR2 抗体进行活细胞成像研究表明，VEGFR2 通过早期和晚期内体运输到达内溶酶体，在此处 VEGFR2 被降解。
我们的研究表明，在 VEGF-A 结合和内化后，未占据的 VEGFR1 可能会循环到细胞表面，从而使其能够重新利用，而大多数内化的 VEGFR2 则被靶向降解。
