Venous malformations (VMs), most of which associated with activating mutations in the endothelial cells (ECs) tyrosine kinase receptor TIE2, are characterized by dilated and immature veins with scarce smooth muscle cells (SMCs) coverage. However, the underlying mechanism of interaction between ECs and SMCs responsible for VMs has not been fully understood. Here, we screened 5 patients with TIE2-L914F mutation who were diagnosed with VMs by SNP sequencing, and we compared the expression of platelet-derived growth factor beta (PDGFB) and α-SMA in TIE2 mutant veins and normal veins by immunohistochemistry. In vitro, we generated TIE2-L914F-expressing human umbilical vein endothelial cells (HUVECs) and performed BrdU, CCK-8, transwell and tube formation experiments on none-transfected and transfected ECs. Then we investigated the effects of rapamycin (RAPA) on cellular characteristics. Next we established a co-culture system and investigated the role of AKT/FOXO1/PDGFB in regulating cross-talking of mutant ECs and SMCs. VMs with TIE2-L914F mutation showed lower expression of PDGFB and α-SMA than normal veins. TIE2 mutant ECs revealed enhanced cell viability and motility, and decreased tube formation, whereas these phenotypes could be reversed by rapamycin. Mechanically, RAPA ameliorated the physiological function of mutant ECs by inhibiting AKT-mTOR pathway, but also facilitated the nuclear location of FOXO1 and the expression of PDGFB in mutant ECs, and then improved paracrine interactions between ECs and SMCs. Moreover, TIE2 mutant ECs strongly accelerated the transition of SMCs from contractile phenotype to synthetic phenotype, whereas RAPA could prevent the phenotype transition of SMCs. Our data demonstrate a previously unknown mechanistic linkage of AKT-mTOR/FOXO1 pathway between mutant ECs and SMCs in modulating venous dysmorphogenesis, and AKT/FOXO1 axis might be a potential therapeutic target for the recovery of TIE2-mutation causing VMs.

中文翻译：

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AKT/FOXO1 轴将 TIE2 突变的静脉畸形中内皮细胞和周细胞的串扰联系起来。

静脉畸形 (VM)，其中大部分与内皮细胞 (EC) 酪氨酸激酶受体 TIE2 中的激活突变有关，其特征是扩张和未成熟的静脉，平滑肌细胞 (SMC) 覆盖稀少。然而，负责 VM 的 EC 和 SMC 之间相互作用的潜在机制尚未完全了解。在这里，我们筛选了 5 例通过 SNP 测序诊断为 VMs 的 TIE2-L914F 突变患者，并通过免疫组织化学比较了 TIE2 突变静脉和正常静脉中血小板衍生生长因子β（PDGFB）和 α-SMA 的表达。在体外，我们生成了表达 TIE2-L914F 的人脐静脉内皮细胞 (HUVEC)，并对未转染和转染的 EC 进行了 BrdU、CCK-8、transwell 和管形成实验。然后我们研究了雷帕霉素 (RAPA) 对细胞特性的影响。接下来我们建立了一个共培养系统，并研究了 AKT/FOXO1/PDGFB 在调节突变 ECs 和 SMCs 串扰中的作用。具有 TIE2-L914F 突变的 VM 显示出比正常静脉更低的 PDGFB 和 α-SMA 表达。TIE2 突变的 ECs 显示细胞活力和运动性增强，管形成减少，而这些表型可以被雷帕霉素逆转。从机制上讲，RAPA 通过抑制 AKT-mTOR 通路改善了突变 ECs 的生理功能，但也促进了 FOXO1 的核定位和突变 ECs 中 PDGFB 的表达，进而改善了 ECs 和 SMC 之间的旁分泌相互作用。此外，TIE2 突变的 ECs 强烈加速了 SMCs 从收缩表型到合成表型的转变，而 RAPA 可以阻止 SMC 的表型转变。我们的数据证明了突变 EC 和 SMC 之间的 AKT-mTOR/FOXO1 通路在调节静脉畸形发生中的先前未知的机械联系，并且 AKT/FOXO1 轴可能是恢复导致 TIE2 突变的 VM 的潜在治疗靶点。