Abstract
Receptor-mediated transcytosis (RMT) is a principal pathway for transport of macromolecules essential for brain function across the blood–brain barrier (BBB). Antibodies or peptide ligands which bind RMT receptors are often co-opted for brain delivery of biotherapeutics. Constitutively recycling transferrin receptor (TfR) is a prototype receptor utilized to shuttle therapeutic cargos across the BBB. Several other BBB-expressed receptors have been shown to mediate transcytosis of antibodies or protein ligands including insulin receptor (INSR) and insulin-like growth factor-1 receptor (IGF1R), lipid transporters LRP1, LDLR, LRP8 and TMEM30A, solute carrier family transporter SLC3A2/CD98hc and leptin receptor (LEPR). In this study, we analyzed expression patterns of genes encoding RMT receptors in isolated brain microvessels, brain parenchyma and peripheral organs of the mouse and the human using RNA-seq approach. IGF1R, INSR and LRP8 were highly enriched in mouse brain microvessels compared to peripheral tissues. In human brain microvessels only INSR was enriched compared to either the brain or the lung. The expression levels of SLC2A1, LRP1, IGF1R, LRP8 and TFRC were significantly higher in the mouse compared to human brain microvessels. The protein expression of these receptors analyzed by Western blot and immunofluorescent staining of the brain microvessels correlated with their transcript abundance. This study provides a molecular transcriptomics map of key RMT receptors in mouse and human brain microvessels and peripheral tissues, important to translational studies of biodistribution, efficacy and safety of antibodies developed against these receptors.
Highlights
The receptor-mediated transcytosis (RMT) is a vesicular transcellular route by which various macromolecules are transported across a barrier, typically formed by a cell monolayer [1, 2]
brain microvessels (BMV) exhibited a strong immunoreactivity for CD31/PECAM-1 (Fig. 1a, c), continuous staining with the abluminal marker collagen IV, as well as a weak and ‘spotty’ immunoreactivity for glial fibrillary acidic protein (GFAP) (Fig. 1e, g)
Endothelial cell markers coagulation factor VIII-related antigen (F8), E-selectin (Sele), VE-cadherin (Cdh5) and Pecam1/CD31 showed 4 to eightfold enrichment in BMV preparations compared to vessel-depleted brain parenchyma; brain-endothelial cell-specific Slc2a1/glucose transporter (Glut1) showed over 60-fold enrichment in BMVs
Summary
The receptor-mediated transcytosis (RMT) is a vesicular transcellular route by which various macromolecules are transported across a barrier, typically formed by a cell monolayer [1, 2]. A ligand binding to a receptor on the luminal surface of brain endothelial cells (BEC) triggers ligand-receptor complex endocytosis, routing through various intracellular endosomal compartments where cargo is detached from the receptor and released on the abluminal side, while the receptor recycles ‘back’ to accept additional cargo molecules [7,8,9,10,11] This pathway has been well described for the transferrin receptor (TfR), which undergoes constitutive recycling and has been considered a ‘prototypical’ trigger of the RMT pathway [7,8,9,10,11,12,13]. Whereas the mechanisms of antibody transport by some of these targets, in particular SLC family of transporters, is not fully understood, for the purpose of this manuscript they will be considered in comparison with ‘classical’ RMT-triggered pathways
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