PURPOSE: The expression level of the transferrin receptor (TfR) on brain capillary endothelial cells (BCECs) and the endocytosis of 125I-transferrin (125I-Tf) by this receptor was investigated. Furthermore, the influence of iron, the iron scavenger deferoxamine mesylate (DFO), astrocytic factors, a GTP-ase inhibitor (tyrphostin-A8, T8), lipopolysaccharide (LPS), and the radical scavenger N-acetyl-L-cysteine (NAC) on the TfR expression was studied to gain insight in the use and optimization of the TfR for drug targeting to the brain. METHODS: Experiments were performed with primary cultured bovine BCECs that were incubated with 125I-Tf at 4 degrees C (to determine binding) or at 37 degrees C (to determine endocytosis) in the absence or presence of the modulators. For full saturation curves in the absence or presence of iron or DFO, analysis was performed with a population approach using NONMEM, allowing us to estimate a single value for affinity (Kd, concentration of 50% receptor occupancy) and separate values for maximum receptor occupancy (B(max). RESULTS: On BCECs, the TfR is expressed extracellularly (B(max) of 0.13 fmol/microg cell protein), but also has a large intracellular pool (total B(max) of 1.37 fmol/microg cell protein), and is actively endocytosing Tf via clathrin-coated vesicles. At 4 degrees C, a Kd of 2.38 microg/ml was found, whereas the Kd at 37 degrees C was 5.03 microg/ml. Furthermore, DFO is able to increase both the extracellular as well as the total binding capacity to 0.63 and 3.67 fmol/microg cell protein, respectively, whereas it had no influence on Kd. B(max) at 37 degrees C after DFO preincubation was also increased from 0.90 to 2.31 fmol/microg cell protein. Other modulators had no significant influence on the TfR expression levels, though LPS increased cellular protein concentrations after 2-h preincubation. CONCLUSIONS: The TfR is expressed on BCECs and actively endocytoses Tf, making it a suitable target for drug delivery to the bloodbrain barrier and the CNS. DFO up-regulates the TfR expression level, which may influence targeting efficiency.
PURPOSE: The expression level of the transferrin receptor (TfR) on brain capillary endothelial cells (BCECs) and the endocytosis of 125I-transferrin (125I-Tf) by this receptor was investigated. Furthermore, the influence of iron, the iron scavenger deferoxamine mesylate (DFO), astrocytic factors, a GTP-ase inhibitor (tyrphostin-A8, T8), lipopolysaccharide (LPS), and the radical scavenger N-acetyl-L-cysteine (NAC) on the TfR expression was studied to gain insight in the use and optimization of the TfR for drug targeting to the brain. METHODS: Experiments were performed with primary cultured bovine BCECs that were incubated with 125I-Tf at 4 degrees C (to determine binding) or at 37 degrees C (to determine endocytosis) in the absence or presence of the modulators. For full saturation curves in the absence or presence of iron or DFO, analysis was performed with a population approach using NONMEM, allowing us to estimate a single value for affinity (Kd, concentration of 50% receptor occupancy) and separate values for maximum receptor occupancy (B(max). RESULTS: On BCECs, the TfR is expressed extracellularly (B(max) of 0.13 fmol/microg cell protein), but also has a large intracellular pool (total B(max) of 1.37 fmol/microg cell protein), and is actively endocytosing Tf via clathrin-coated vesicles. At 4 degrees C, a Kd of 2.38 microg/ml was found, whereas the Kd at 37 degrees C was 5.03 microg/ml. Furthermore, DFO is able to increase both the extracellular as well as the total binding capacity to 0.63 and 3.67 fmol/microg cell protein, respectively, whereas it had no influence on Kd. B(max) at 37 degrees C after DFO preincubation was also increased from 0.90 to 2.31 fmol/microg cell protein. Other modulators had no significant influence on the TfR expression levels, though LPS increased cellular protein concentrations after 2-h preincubation. CONCLUSIONS: The TfR is expressed on BCECs and actively endocytoses Tf, making it a suitable target for drug delivery to the bloodbrain barrier and the CNS. DFO up-regulates the TfR expression level, which may influence targeting efficiency.
Authors: Hans C Helms; N Joan Abbott; Malgorzata Burek; Romeo Cecchelli; Pierre-Olivier Couraud; Maria A Deli; Carola Förster; Hans J Galla; Ignacio A Romero; Eric V Shusta; Matthew J Stebbins; Elodie Vandenhaute; Babette Weksler; Birger Brodin Journal: J Cereb Blood Flow Metab Date: 2016-02-11 Impact factor: 6.200
Authors: James A Duce; Andrew Tsatsanis; Michael A Cater; Simon A James; Elysia Robb; Krutika Wikhe; Su Ling Leong; Keyla Perez; Timothy Johanssen; Mark A Greenough; Hyun-Hee Cho; Denise Galatis; Robert D Moir; Colin L Masters; Catriona McLean; Rudolph E Tanzi; Roberto Cappai; Kevin J Barnham; Giuseppe D Ciccotosto; Jack T Rogers; Ashley I Bush Journal: Cell Date: 2010-09-17 Impact factor: 41.582
Authors: Jason Papademetriou; Carmen Garnacho; Daniel Serrano; Tridib Bhowmick; Edward H Schuchman; Silvia Muro Journal: J Inherit Metab Dis Date: 2012-09-12 Impact factor: 4.982