Therapy targeting the neurovascular unit may provide effective neuroprotection against neonatal hypoxia–ischemia (HI). We hypothesized that the peripheral injection of hypoxia-preconditioned human umbilical vein endothelial cells (HUVECs) following HI protects against neurovascular damage and provides long-term neuroprotection in a postpartum (P) day-7 rat pup model. Compared with normoxic HUVECs, hypoxic HUVECs showed enhanced migration and angiogenesis in vitro and had augmented migration effects into the brain when administered intraperitoneally in vivo after HI. Moreover, 24 and 72 h post-HI, the hypoxic HUVECs group but not the normoxic HUVECs or culture-medium groups had significantly higher preservation of microvessels and neurons, and attenuation of blood–brain barrier damage than the normal-saline group. Compared to control or normal-saline groups, only the hypoxic HUVECs group had no impaired foot steps and showed a significant reduction of brain area loss at P42. Next-generation sequencing showed hypoxia-induced upregulation and downregulation of 209 and 215 genes in HUVECs, respectively. Upstream regulator analysis by ingenuity pathway analysis (IPA) identified hypoxia-inducible factor 1-alpha as the key predicted activated transcription regulator. After hypoxia, 12 genes (ADAMTS1, EFNA1, HIF1A, LOX, MEOX2, SELE, VEGFA, VEGFC, CX3CL1, HMMR, SDC, and SERPINE) associated with migration and/or angiogenesis were regulated in HUVECs. In addition, 6 genes (VEGFA, VEGFC, NTN4, TGFA, SERPINE1, and CX3CL1) involved in the survival of endothelial and neuronal cells were also markedly altered in hypoxic HUVECs. Thus, cell therapy by using hypoxic HUVECs that enhance migration and neurovascular protection may provide an effective therapeutic strategy for treating neonatal asphyxia.
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