Figure 3. Influence of cellular and secreted factors on formation of CC-like fenestrated endothelium.

(a) Schematic representation of experimental design and conditions. i) RPE monolayers (“RPE”) cultured on the transwell membrane in routine culture media (RDM) medium, ii) MSC monolayers (“MSC”) cultured on the transwell membrane in routine MSC culture medium, iii) EC-MSC co-cultures in RDM media apically and routine EC culture media of the transwell (“EC-MSC”) and iv) EC-MSC co-cultures treated apically with RPE-CM (“(RPE-CM)-CC” and basally with EC media.

(b-d) Quantitative ELISA analyses of VEGF-A for media collected from the (b) apical or (c, d) basal chambers of the transwell. Data normalized to (b, c) RPE and (d) (RPE-CM)-CC. n = 3 biological replicates. * p ≤ 0.05; *** p ≤ 0.001.

(e-g) Quantitative ELISA analyses of TGF-ß1 for medium collected from the (e) apical and (f, g) basal chambers of the transwell. Data normalized to (e, f) RPE and (g) (RPE-CM)-CC. n= 3 biological replicates. * p ≤ 0.05; *** p ≤ 0.001.

(h) Schematic representation of experimental set-up utilized to evaluate the impact of VEGF-A or TGF-ß1 inhibition using daily supplementation of neutralizing antibody αVEGF-A:10μg/ml; αTGF-ß1: 10μg/ml) on development of PLVAP-positive vascular networks in the (RPE-CM)-CC model.

(i, j) Immunofluorescence images (i) and quantitative analyses (j) showing CD31 (green), PLVAP (red), and DAPI (blue) expression in untreated (RPE-CM)-CC or (RPE-CM)-CC treated with neutralizing antibodies for VEGF-A (“αVEGF-A”; 10μg/ml) or TGF-ß1 (“αTGF-ß1”; 10μg/ml) at the day 7 time point. Of note, day 7 timepoint was chosen for this analysis as abundant amount of PLVAP-positive vascular networks were consistently observed by this timepoint in the CC model. The total number of PLVAP⁺ tubes were normalized to untreated (RPE-CM)-CC. n = 3 biological replicates. *** p ≤ 0.001. Scale bars = 20 μm.

See also Table S2.