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BackgroundThe objectives of the present study were to investigate whether the expression of transferrin receptor 1 (TfR1), glucose transporter 1 (Glut1), or Cluster of Differentiation 98 Heavy Chain (CD98hc) is epigenetically regulated in brain capillary endothelial cells (BCECs) denoting the blood-brain barrier (BBB).MethodsThe expression of these targets was investigated both in vitro and in vivo following treatment with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). Mice were injected intraperitoneally with VPA followed by analysis of isolated brain capillaries, and the capillary depleted brain samples. Brain tissue, isolated brain capillaries, and cultured primary endothelial cells were analyzed by RT-qPCR, immunolabeling and ELISA for expression of TfR1, Glut1 and CD98hc. We also studied the vascular targeting in VPA-treated mice injected with monoclonal anti-transferrin receptor (Ri7) conjugated with 1.4 nm gold nanoparticles. ResultsValidating the effects of VPA on gene transcription in BCECs, transcriptomic analysis identified 24,371 expressed genes, of which 305 were differentially expressed with 192 upregulated and 113 downregulated genes. In vitro using BCECs co-cultured with glial cells, the mRNA expression of Tfrc was significantly higher after VPA treatment for 6 h with its expression returning to baseline after 24 h. Conversely, the mRNA expression of Glut1 and Cd98hc was unaffected by VPA treatment. In vivo, the TfR1 protein expression in brain capillaries increased significantly after treatment with both 100 mg/kg and 400 mg/kg VPA. Conversely, VPA treatment did not increase GLUT1 or CD98hc. Using ICP-MS-based quantification, the brain uptake of nanogold conjugated anti-TfR1 antibodies was non-significant in spite of increased expression of TfR1. ConclusionsWe report that VPA treatment upregulates TfR1 at the BBB both in vivo and in vitro in isolated primary endothelial cells. In contrast, VPA treatment does not influence the expression of GLUT1 and CD98hc. The increase in the overall TfR1 protein expression however does not increase transport of TfR-targeted monoclonal antibody and indicates that targeted delivery using the transferrin receptor should aim for increased mobilization of already available transferrin receptor molecules to improve trafficking through the BBB. |
