In:
BioMed Research International, Hindawi Limited, Vol. 2019 ( 2019-09-10), p. 1-12
Abstract:
Background and Aims . Vascular endothelial growth factor (VEGF) receptors (VEGFR1 and VEGFR2) bind VEGF-A with high affinity. This study sought to determine the relative contributions of these two receptors to receptor-mediated endocytosis of VEGF-A and to clarify their endocytic itineraries in rat liver sinusoidal endothelial cells (LSECs). Methods . Isolated LSECs and radiolabeled VEGF-A were used to examine surface binding and receptor-mediated endocytosis. Quantitative real time RT-PCR (Q-RT-PCR) and Western blotting were applied to demonstrate receptor expression. Results . Q-RT-PCR analysis showed that VEGFR1 and VEGFR2 mRNA were expressed in LSECs. Ligand saturation analysis at 4°C indicated two different classes of [ 125 I]-VEGFA binding sites on LSECs with apparent dissociation constants of 8 and 210 pM. At 37°C, LSECs efficiently took up and degraded [ 125 I]-VEGF-A for at least 2 hours. Uptake of [ 125 I]-VEGF-A by LSECs was blocked by dynasore that inhibits dynamin-dependent internalization, whereas inhibition of cysteine proteases by leupeptin inhibited degradation without affecting the uptake of [ 125 I]-VEGF-A, suggesting that it is degraded following transport to lysosomes. Incubation of LSECs in the continued presence of a saturating concentration of unlabeled VEGF-A at 37°C was associated with a loss of as much as 75% of the total VEGFR2 within 30 min as shown by Western blot analysis, whereas there was no appreciable decrease in protein levels for VEGFR1 after 120 min incubation, suggesting that VEGF-A stimulation downregulates VEGFR2, but not VEGFR1, in LSECs. This possibility was supported by the observation that a hexapeptide that specifically blocks VEGF-A binding to VEGFR1 caused a marked reduction in the uptake of [ 125 I]-VEGF-A, whereas a control peptide had no effect. Finally, live cell imaging studies using a fluorescently labeled anti-VEGFR2 antibody showed that VEGFR2 was transported via early and late endosomes to reach endolysosomes where degradation of the VEGFR2 takes place. Conclusion . Our studies suggest that, subsequent to VEGF-A binding and internalization, the unoccupied VEGFR1 may recycle to the cell surface allowing its reutilization, whereas the majority of the internalized VEGFR2 is targeted for degradation.
Type of Medium:
Online Resource
ISSN:
2314-6133
,
2314-6141
DOI:
10.1155/2019/5496197
Language:
English
Publisher:
Hindawi Limited
Publication Date:
2019
detail.hit.zdb_id:
2698540-8
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