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Receptor-Mediated Endocytosis of VEGF-A in Rat Liver Sinusoidal Endothelial Cells
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 l...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Hindawi
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754870/ https://www.ncbi.nlm.nih.gov/pubmed/31583245 http://dx.doi.org/10.1155/2019/5496197 |
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author | Mousavi, Seyed Ali Skjeldal, Frode Fønhus, Marita Sporstøl Haugen, Linda Hofstad Eskild, Winnie Berg, Trond Bakke, Oddmund |
author_facet | Mousavi, Seyed Ali Skjeldal, Frode Fønhus, Marita Sporstøl Haugen, Linda Hofstad Eskild, Winnie Berg, Trond Bakke, Oddmund |
author_sort | Mousavi, Seyed Ali |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6754870 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Hindawi |
record_format | MEDLINE/PubMed |
spelling | pubmed-67548702019-10-03 Receptor-Mediated Endocytosis of VEGF-A in Rat Liver Sinusoidal Endothelial Cells Mousavi, Seyed Ali Skjeldal, Frode Fønhus, Marita Sporstøl Haugen, Linda Hofstad Eskild, Winnie Berg, Trond Bakke, Oddmund Biomed Res Int Research Article 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. Hindawi 2019-09-10 /pmc/articles/PMC6754870/ /pubmed/31583245 http://dx.doi.org/10.1155/2019/5496197 Text en Copyright © 2019 Seyed Ali Mousavi et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Mousavi, Seyed Ali Skjeldal, Frode Fønhus, Marita Sporstøl Haugen, Linda Hofstad Eskild, Winnie Berg, Trond Bakke, Oddmund Receptor-Mediated Endocytosis of VEGF-A in Rat Liver Sinusoidal Endothelial Cells |
title | Receptor-Mediated Endocytosis of VEGF-A in Rat Liver Sinusoidal Endothelial Cells |
title_full | Receptor-Mediated Endocytosis of VEGF-A in Rat Liver Sinusoidal Endothelial Cells |
title_fullStr | Receptor-Mediated Endocytosis of VEGF-A in Rat Liver Sinusoidal Endothelial Cells |
title_full_unstemmed | Receptor-Mediated Endocytosis of VEGF-A in Rat Liver Sinusoidal Endothelial Cells |
title_short | Receptor-Mediated Endocytosis of VEGF-A in Rat Liver Sinusoidal Endothelial Cells |
title_sort | receptor-mediated endocytosis of vegf-a in rat liver sinusoidal endothelial cells |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754870/ https://www.ncbi.nlm.nih.gov/pubmed/31583245 http://dx.doi.org/10.1155/2019/5496197 |
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