Clinic Center of Human Gene Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Ave., Wuhan, 430022, China.
Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
J Transl Med. 2022 Dec 28;20(1):627. doi: 10.1186/s12967-022-03841-6.
Angiogenesis is involved in many pathological and physiological processes and is mainly driven by hypoxia. Karyopherin subunit alpha 2 (KPNA2), a member of the nuclear transport protein family, was recently shown to be induced by hypoxia in various types of tumours, so we aimed to investigate the role and mechanism of KPNA2 in angiogenesis under hypoxia.
After overexpression or knockdown of KPNA2 in human umbilical vein endothelial cells (HUVEC) by adenovirus vector infection, the tube formation, proliferation and migration of HUVEC under hypoxia were detected by tubule formation assay, 5-ethynyl-2'-deoxyuridine (EdU) staining and Transwell assay, respectively. After overexpression or knockdown of KPNA2 in a murine hindlimb ischemia model by local injection of purified adenovirus vector into the gastrocnemius muscle, blood flow changes were examined with a laser Doppler system. Changes in KPNA2-binding proteins under hypoxia were detected by immunoprecipitation-mass spectrometry (IP-MS) and co-immunoprecipitation (Co-IP). The effect of KPNA2 on signal transducer and activator of transcription 3 (STAT3) was detected by Western blotting and quantitative RT‒PCR.
KPNA2 was upregulated in the HUVEC hypoxia model and murine hindlimb ischemia model. Overexpression of KPNA2 increased the proliferation, migration and tube formation of HUVEC under hypoxia, while knockdown of KPNA2 reduced the proliferation, migration and tube formation of HUVEC. Overexpression of KPNA2 promoted the restoration of blood flow in the murine hindlimb ischemia model, while knockout of KPNA2 inhibited the restoration of blood flow in the murine hindlimb ischemia model. Mechanistically, hypoxia promoted the binding of STAT3 to KPNA2. Overexpression of KPNA2 promoted STAT3 phosphorylation and then upregulated vascular endothelial growth factor (VEGF) and angiopoietin 2(ANGPT2), whereas knockdown of KPNA2 inhibited STAT3 phosphorylation and then downregulated VEGF and ANGPT2.
Our study demonstrates that hypoxia promotes the binding of STAT3 to KPNA2 and KPNA2 promotes angiogenesis under hypoxia by promoting the binding of STAT3 and JAK1 and regulating STAT3 phosphorylation.
血管生成参与许多病理和生理过程，主要由缺氧驱动。核转运蛋白家族的成员核孔蛋白亚单位 α2（KPNA2）最近在各种类型的肿瘤中被证明是由缺氧诱导的，因此我们旨在研究 KPNA2 在缺氧下对血管生成的作用和机制。
通过腺病毒载体感染过表达或敲低人脐静脉内皮细胞（HUVEC）中的 KPNA2 后，通过小管形成、5-乙炔基-2'-脱氧尿苷（EdU）染色和 Transwell 测定分别检测 HUVEC 在缺氧下的管形成、增殖和迁移。通过局部注射纯化的腺病毒载体到腓肠肌中，在小鼠后肢缺血模型中过表达或敲低 KPNA2 后，用激光多普勒系统检查血流变化。通过免疫沉淀-质谱（IP-MS）和共免疫沉淀（Co-IP）检测缺氧下 KPNA2 结合蛋白的变化。通过 Western blot 和定量 RT-PCR 检测 KPNA2 对信号转导和转录激活因子 3（STAT3）的影响。
KPNA2 在 HUVEC 缺氧模型和小鼠后肢缺血模型中上调。过表达 KPNA2 增加了缺氧下 HUVEC 的增殖、迁移和管形成，而敲低 KPNA2 减少了缺氧下 HUVEC 的增殖、迁移和管形成。过表达 KPNA2 促进了小鼠后肢缺血模型中血流的恢复，而敲除 KPNA2 抑制了小鼠后肢缺血模型中血流的恢复。机制上，缺氧促进了 STAT3 与 KPNA2 的结合。过表达 KPNA2 促进了 STAT3 的磷酸化，进而上调了血管内皮生长因子（VEGF）和血管生成素 2（ANGPT2），而敲低 KPNA2 抑制了 STAT3 的磷酸化，进而下调了 VEGF 和 ANGPT2。
我们的研究表明，缺氧促进了 STAT3 与 KPNA2 的结合，而 KPNA2 通过促进 STAT3 和 JAK1 的结合以及调节 STAT3 磷酸化来促进缺氧下的血管生成。
