Figure 3.
Differential VEGF Receptor Localization in Microvascular ECs in Brain and Retina
(A and B) Cryoimmunogold EM analysis of VEGFR1 and -R2 in primary rat brain MVECs (A) or mouse hippocampal microvessels (B) showed predominant apical/luminal (black) and basal/abluminal (brown) localization, respectively. Mean distribution of each VEGF receptor (±SEM) was determined by quantifying gold particles located within ca. 20 nm of the plasma membrane (as indicated by arrowheads) in five independent sections, each comprising at least 10 μm of continuous plasma membrane. The scale bars represent 100 nm.
(C) VEGF receptor distribution was also analyzed in postconfluent rat cerebral MVECs by biotinylation of either the apical or, in the presence of EDTA, the apical and basal membranes (ap+bl). Biotinylated proteins were isolated and analyzed by western blot. Shown are representative immunoblots of VEGFR1 and VEGFR2 and quantitative distribution analysis from three experiments (means ± SEM).
(D) VEGFR1 but not -R2 antibodies bound to the retinal vasculature of mice within 5 min of luminal delivery through cardiac injection. However, in the same animals, both VEGFR1 and -R2 antibodies were found bound to alveolar microvessels in the lung. Inversely, when unfixed, nonpermeabilized retinae were incubated with VEGFR antibodies (abluminal), only VEGFR2 was found to stain microvessels significantly. All whole mounts and sections were counterstained with the vessel marker isolectin B4 and analyzed by confocal microcopy. Shown are optical sections of ca. 8 μm thickness of the retinal ganglion cell layer or the center of the lung. The scale bars represent 20 μm.