TY - JOUR
T1 - Quantitative Assessment of the Apical and Basolateral Membrane Expression of VEGFR2 and NRP2 in VEGF-A-stimulated Cultured Human Umbilical Vein Endothelial Cells
AU - Bosma, Esmeralda K.
AU - Darwesh, Shahan
AU - Zheng, Jia Y.
AU - van Noorden, Cornelis J. F.
AU - Schlingemann, Reinier O.
AU - Klaassen, Ingeborg
N1 - Funding Information: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was made possible by grants through AMC Foundation (to IK and ROS) and the Slovenian Research Agency (research projects P1-0245 and J3-2526 to CJFVN). Publisher Copyright: © The Author(s) 2022.
PY - 2022/8/1
Y1 - 2022/8/1
N2 - Endothelial cells (ECs) form a precisely regulated polarized monolayer in capillary walls. Vascular endothelial growth factor-A (VEGF-A) induces endothelial hyperpermeability, and VEGF-A applied to the basolateral side, but not the apical side, has been shown to be a strong barrier disruptor in blood–retinal barrier ECs. We show here that VEGF-A presented to the basolateral side of human umbilical vein ECs (HUVECs) induces higher permeability than apical stimulation, which is similar to results obtained with bovine retinal ECs. We investigated with immunocytochemistry and confocal imaging the distribution of VEGF receptor-2 (VEGFR2) and neuropilin-2 (NRP2) in perinuclear apical and basolateral membrane domains. Orthogonal z-sections of cultured HUVECs were obtained, and the fluorescence intensity at the apical and basolateral membrane compartments was measured. We found that VEGFR2 and NRP2 are evenly distributed throughout perinuclear apical and basolateral membrane compartments in unstimulated HUVECs grown on Transwell inserts, whereas basolateral VEGF-A stimulation induces a shift toward basolateral VEGFR2 and NRP2 localization. When HUVECs were grown on coverslips, the distribution of VEGFR2 and NRP2 across the perinuclear apical and basolateral membrane domains was different. Our findings demonstrate that HUVECs dynamically regulate VEGFR2 and NRP2 localization on membrane microdomains, depending on growth conditions and the polarity of VEGF-A stimulation.
AB - Endothelial cells (ECs) form a precisely regulated polarized monolayer in capillary walls. Vascular endothelial growth factor-A (VEGF-A) induces endothelial hyperpermeability, and VEGF-A applied to the basolateral side, but not the apical side, has been shown to be a strong barrier disruptor in blood–retinal barrier ECs. We show here that VEGF-A presented to the basolateral side of human umbilical vein ECs (HUVECs) induces higher permeability than apical stimulation, which is similar to results obtained with bovine retinal ECs. We investigated with immunocytochemistry and confocal imaging the distribution of VEGF receptor-2 (VEGFR2) and neuropilin-2 (NRP2) in perinuclear apical and basolateral membrane domains. Orthogonal z-sections of cultured HUVECs were obtained, and the fluorescence intensity at the apical and basolateral membrane compartments was measured. We found that VEGFR2 and NRP2 are evenly distributed throughout perinuclear apical and basolateral membrane compartments in unstimulated HUVECs grown on Transwell inserts, whereas basolateral VEGF-A stimulation induces a shift toward basolateral VEGFR2 and NRP2 localization. When HUVECs were grown on coverslips, the distribution of VEGFR2 and NRP2 across the perinuclear apical and basolateral membrane domains was different. Our findings demonstrate that HUVECs dynamically regulate VEGFR2 and NRP2 localization on membrane microdomains, depending on growth conditions and the polarity of VEGF-A stimulation.
KW - apicobasal
KW - endothelial barrier
KW - membrane microdomains
KW - receptors
KW - vascular endothelial growth factor A
KW - vascular endothelial growth factor receptor
UR - http://www.scopus.com/inward/record.url?scp=85134743200&partnerID=8YFLogxK
U2 - https://doi.org/10.1369/00221554221115767
DO - https://doi.org/10.1369/00221554221115767
M3 - Article
C2 - 35876388
SN - 0022-1554
VL - 70
SP - 557
EP - 569
JO - Journal of Histochemistry and Cytochemistry
JF - Journal of Histochemistry and Cytochemistry
IS - 8
ER -