[Effects of exosomes derived from normoxic or hypoxic human umbilical vein endothelial cells on hypoxia-induced cardiomyocyte injury and cardiac fibroblast activation in vitro].

Abstract

Objectives: To investigate the effects of exosomes derived from human umbilical venous endothelial cells (HUVECs) cultured in normoxic and hypoxic conditions (N-exo and H-exo, respectively) on hypoxia-induced injury of human cardiomyocyte AC16 cells and activation of rat cardiac fibroblasts (RCFs) in vitro.

Methods: N-exo and H-exo were extracted from HUVECs by differential centrifugation and sucrose density gradient centrifugation and characterized. AC16 cells and RCFs in normoxic culture or hypoxic culture (1% O₂ for 24 h) were treated with N-exo or H-exo, and the changes in protein and mRNA levels were examined with Western blotting and RT-qPCR. Intracellular ROS levels and apoptosis in AC16 cells were assessed by DCFH-DA staining and TUNEL staining, and the changes in proliferation of RCFs were evaluated with EdU assay.

Results: N-exo and H-exo with densities of 1.5×10⁹ and 7.2×10⁸ mL^-1 were obtained, respectively, which showed abundant protein components and expressed the exosomal marker HSP70. In AC16 cells, hypoxic exposure significantly upregulated CK-MB mRNA and downregulated Cx43 and GPX4 mRNA levels, causing also enhanced expressions of ALP, IL-1β and cTnT proteins and increased intracellular ROS content and cell apoptosis. These changes were effectively alleviated by treatment with 10⁷ mL^-1 N-exo but worsened after treatment with H-exo. In RCFs, hypoxia significantly increased the expressions of collagen I, collagen III and α‑SMA and cell proliferation, which were suppressed by N-exo treatment but further enhanced by H-exo treatment. Hypoxia exposure increased HIF-1α and VEGF expression and phosphorylation level of AKT in both AC16 cells and RCFs; N-exo obviously inhibited while H-exo further enhanced these changes. Px-478, a specific HIF-1α inhibitor, significantly inhibited hypoxia-induced AC16 cell injury and RCF activation.

Conclusions: N-exo derived from HUVECs inhibits while H-exo exacerbates hypoxia-induced AC16 cell injury and RCF activation possibly via the HIF-1α pathway.