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. 1999 Aug;155(2):421-8.
doi: 10.1016/S0002-9440(10)65138-3.

Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris. Evidence for a trophic paracrine relation

H G Blaauwgeers  1 , G M Holtkamp, H Rutten, A N Witmer, P Koolwijk, T A Partanen, K Alitalo, M E Kroon, A Kijlstra, V W van Hinsbergh, R O Schlingemann
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Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris. Evidence for a trophic paracrine relation

H G Blaauwgeers et al. Am J Pathol. 1999 Aug.

Abstract

The retinal pigment epithelium (RPE) maintains the choriocapillaris (CC) in the normal eye and is involved in the pathogenesis of choroidal neovascularization in age-related macular degeneration. Vascular endothelial growth factor-A (VEGF) is produced by differentiated human RPE cells in vitro and in vivo and may be involved in paracrine signaling between the RPE and the CC. We investigated whether there is a polarized secretion of VEGF by RPE cells in vitro. Also, the localization of VEGF receptors in the human retina was investigated. We observed that highly differentiated human RPE cells, cultured on transwell filters in normoxic conditions, produced two- to sevenfold more VEGF toward their basolateral side as compared to the apical side. In hypoxic conditions, VEGF-A secretion increased to the basal side only, resulting in a three- to 10-fold higher basolateral secretion. By immunohistochemistry in 30 human eyes and in two cynomolgus monkey eyes, KDR (VEGFR-2) and flt-4 (VEGFR-3) were preferentially localized at the side of the CC endothelium facing the RPE cell layer, whereas flt-1 (VEGFR-1) was found on the inner CC and on other choroidal vessels. Our results indicate that RPE secretes VEGF toward its basal side where its receptor KDR is located on the adjacent CC endothelium, suggesting a role of VEGF in a paracrine relation, possibly in cooperation with flt-4 and its ligand. This can explain the known trophic function of the RPE in the maintenance of the CC and its fenestrated permeable phenotype and points to a role for VEGF in normal eye functioning. Up-regulated basolateral VEGF secretion by RPE in hypoxia or loss of polarity of VEGF production may play a role in the pathogenesis of choroidal neovascularization.

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Figures

Figure 1.
Figure 1.
Transepithelial resistance (TER) (Ohm·cm2 ± SD) of RPE cells of the four donors A, B, C, and D, cultured on transwell filters for 3 weeks (each bar represents 12 filters).
Figure 2.
Figure 2.
Characterization of RPE monolayers cultured on transwell filter for 3 weeks. A: Light microscopy of RPE cell monolayer (donor B), stained with hematoxylin-eosin. B: Electron microscopy of the apical side of the RPE cell monolayer. Note the presence of apical microvilli and between the cells a structure (indicated by arrows) suggesting the presence of a tight junction. C and D: Immunofluorescence microscopy of RPE cell monolayers of donor B (low TER: 35 Ohm·cm2) (C) and donor D (high TER: 100 Ohm·cm2) (D) stained for tight junction-associated protein ZO-1. Note staining for ZO-1 at the intercellular junctions.
Figure 3.
Figure 3.
Total VEGF secreted (basal and apical side together, ± SE of mean) in 24 hours by RPE cells of the four donors A, B, C, and D cultured on transwell filters for 3 weeks (each bar represents three filters).
Figure 4.
Figure 4.
Differential VEGF secretion (± SE of mean) toward the apical and basal sides of transwell filters by RPE cell monolayers of the four donors A, B, C, and D in normal (37°C, 20% O2, 5% CO2, moist environment) and in hypoxic (37°C, 1% O2, 5% CO2, moist environment) conditions in 24 hours (each bar represents three filters). Note that VEGF secretion is higher to the basal side than to the apical side (statistically significant for all conditions, P < 0.05). In hypoxic conditions, VEGF secretion is higher, mainly to the basal side (statistical significance indicated by * = P < 0.05 and ** = P < 0.01).
Figure 5.
Figure 5.
Immunoperoxidase staining of frozen tissue sections of human choroid with monoclonal antibody PAL-E, recognizing fenestrated endothelium (A), and antibodies recognizing VEGFR-1 (flt-1) (B), VEGFR-2 (KDR) (C), and VEGFR-3 (flt-4) (D). Note the intense staining of the entire choriocapillaris and other choroidal vessels for PAL-E (A), in contrast to the localized staining of the choriocapillaris at the side of the RPE cell layer for VEGFR-2 (KDR) (C) and VEGFR-3 (flt-4) (D). VEGFR-1 (flt-1) staining can be observed in the inner CC and in a large vessel in the choroid (B). Staining of vascular structures is indicated by arrows.
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