Exosomes Derived from Glioma Cells under Hypoxia Promote Angiogenesis through Up-regulated Exosomal Connexin 43

Abstract

Glioblastoma multiform (GBM) is a highly aggressive primary brain tumor. Exosomes derived from glioma cells under a hypoxic microenvironment play an important role in tumor biology including metastasis, angiogenesis and chemoresistance. However, the underlying mechanisms remain to be elucidated. In this study, we aimed to explore the role of connexin 43 on exosomal uptake and angiogenesis in glioma under hypoxia. U251 cells were exposed to 3% oxygen to achieve hypoxia, and the expression levels of HIF-1α and Cx43, involved in the colony formation and proliferation of cells were assessed. Exosomes were isolated by differential velocity centrifugation from U251 cells under normoxia and hypoxia (Nor-Exos and Hypo-Exos), respectively. Immunofluorescence staining, along with assays for CCK-8, tube formation and wound healing along with a transwell assay were conducted to profile exosomal uptake, proliferation, tube formation, migration and invasion of HUVECs, respectively. Our results revealed that Hypoxia significantly up-regulated the expression of HIF-1α in U251 cells as well as promoting proliferation and colony number. Hypoxia also increased the level of Cx43 in U251 cells and in the exosomes secreted. The uptake of Dio-stained Hypo-Exos by HUVECs was greater than that of Nor-Exos, and inhibition of Cx43 by 37,43 gap27 or lenti-Cx43-shRNA efficiently prevented the uptake of Hypo-Exos by recipient endothelial cells. In addition, the proliferation and total loops of HUVECs were remarkably increased at 24 h, 48 h, and 10 h after Hypo-Exos, respectively. Notably, 37,43 gap27, a specific Cx-mimetic peptide blocker of Cx37 and Cx43, efficiently alleviated Hypo-Exos-induced proliferation and tube formation by HUVECs. Finally, 37,43 gap27 also significantly attenuated Hypo-Exos-induced migration and invasion of HUVECs. These findings demonstrate that exosomal Cx43 contributes to glioma angiogenesis mediated by Hypo-Exos, and suggests that exosomal Cx43 might serve as a potential therapeutic target for glioblastoma.

Extracellular vesicles as modulators of glioblastoma progression and tumor microenvironment

“…Under 3% oxygen (hypoxia), the level of connexin 43 (Cx43) in exosomes secreted in the glioblastoma cell line U251 cells was elevated. However, compared with PBS (control), exosomal Cx43 remarkably promoted tube formation in human umbilical vein endothelial cells, thereby contributing to angiogenesis in hypoxic glioblastoma TME [ 79 ].…”

Gap Junctions or Hemichannel-Dependent and Independent Roles of Connexins in Fibrosis, Epithelial–Mesenchymal Transitions, and Wound Healing

“…Another study shows that exosomes derived from glioma cells under hypoxia promote the proliferation, tube formation, and migration of human umbilical vein endothelial cells through upregulated exosomal Cx43 [152]. A study by Villamizar et al found that exosomes derived from mesenchymal stem cells may offer a hopeful therapy for patients afflicted with cystic fibrosis (CF).…”

Connexins and Glucose Metabolism in Cancer

“…Seminal work in this area demonstrated that Cx43 was not only present within exosomes, but formed channels that facilitated the transfer of cargo from exosomes into the recipient cells, as well as internalization of the exosomes themselves [ 316 ]. Since this report, roles for connexins within extracellular vesicles is continuing to expand [ 317 , 318 , 319 , 320 , 321 , 322 ]. Regarding cellular bioenergetics, exosomes are capable of altering cancer cell metabolism through the delivery of metabolic pathway intermediates.…”