The Role And Mechanism Of KPNA2 In Angiogenesis

Objective: Angiogenesis involves many pathological and physiological processes,and abnormal angiogenesis can affect the progression of many diseases.Hypoxia is a feature of many ischaemic diseases and tumours and is a strong stimulus for angiogenesis.Nuclear transport protein subunit α2(KPNA2),a member of the nuclear protein transport family,is upregulated by hypoxia in various tumour cells,but the role of KPNA2 in angiogenesis under hypoxia is not clear.The purpose of this paper is to clarify the expression of KPNA2 in a vascular endothelial cell hypoxia model and mouse lower limb ischemia model,to explore the effects of KPNA2 on tube formation,proliferation and migration in vitro and the effect of KPNA2 on mouse lower limb ischemia in vivo,and to find the changes in KPNA2 binding proteins under hypoxic conditions and the mechanism by which KPNA2 promotes angiogenesis.Materials and methods: 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(Ed U)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 Blot and quantitative RT-PCR.Results: The protein expression of KPNA2 increased at 3 hours,6 hours,and 12 hours of hypoxia in umbilical vein endothelial cells.The protein expression of KPNA2 increased on the 3rd day and 7th day in the gastrocnemius muscle of the mouse lower limb ischaemia model.Immunofluorescence showed that KPNA2 partially colocalized with vascular endothelial cells.Overexpression of KPNA2 increased the tube formation,proliferation and migration of HUVECs under hypoxic conditions,whereas knockdown of KPNA2 decreased the tube formation,proliferation and migration of HUVECs.Overexpression of KPNA2 promoted the recovery of blood perfusion and the number of new blood vessels in the mouse lower limb ischaemia model,while knockdown of KPNA2 inhibited the recovery of blood perfusion and the number of new blood vessels in the mouse lower limb ischaemia model.Enrichment analysis of IP-MS proteins showed that KPNA2 was related to VEGF-VEGFR2,and the combination of KPNA2 and STAT3 was increased under hypoxia.KPNA2 overexpression promoted the phosphorylation of STAT3 and increased the m RNA and protein expression of VEGF and ANGPT2.KPNA2 knockdown inhibited the phosphorylation of STAT3 and reduced the m RNA and protein expression of VEGF and ANGPT2.After knockdown of KPNA2,the binding of STAT3 to JAK1 decreased,while the binding to JAK2,TYK2,and SRC remained unchanged.Stattic,an inhibitor of STAT3,reversed the proangiogenic effects of KPNA2.Conclusion: The expression of KPNA2 is increased in an endothelial cell hypoxia model and a mouse lower limb ischaemia model,and there is partial colocalization with vascular endothelial cells in the gastrocnemius muscle of mice with lower limb ischaemia.KPNA2 promotes endothelial cell proliferation,migration and tube formation in vitro and promotes angiogenesis in a mouse lower limb ischaemia model in vivo.Hypoxia promotes the combination of STAT3 and KPNA2,and KPNA2 promotes angiogenesis under hypoxic conditions by promoting the combination of STAT3 and JAK1,regulating STAT3 phosphorylation and upregulating the STAT3 downstream genes VEGF and ANGPT2.