Characterization of isolated porcine RPE cells
Isolated porcine RPE cells showed characteristics of differentiated RPE after 4 weeks of culture on Transwell membranes. Confocal images showed adherens junction protein, β-catenin (Fig. 2a), F-actin (Fig. 2b), and tight junction protein, ZO-1 (Fig. 2c) along the cell-cell junctions, as well as the cytoplasmic expression of the RPE-specific protein, RPE65 (Fig. 2d). The typical RPE cobblestone morphology was also observed in brightfield images of 4-week-old cultures (Fig. 2e). The TEER values increased from 65.38 to 767.05 Ω.cm2 (p = 1.46 E-6) after 4 weeks (Fig. 2f).
Increased strain leads to increased VEGF, IL-6, and IL-8 expression
Finite element analysis showed that the mechanically stressed area’s center experiences the highest strain level (Fig. 3a). These results were compared to the ICC results to determine whether strain levels and increased angiogenic protein expression are correlated.
Confocal images of mechanically-stressed RPE cells showed that in non-stressed cells, F-actin localized to cell-cell junctions (Fig. 2b), while in mechanically stressed cells, the actin filaments distributed diffusely in the cytoplasm (Fig. 3c, h, m). ICC results also showed that the mechanical stress-induced disruption of the F-actin cytoskeleton was associated with increases in VEGF (Fig. 3b-e), IL-6 (Fig. 3g-j), and IL-8 (Fig. 3l-o) expression. The RPE monolayer also began to deform after stretching the Transwell membranes, based on z-stack images and (Fig. 3f, k, p), as predicted by FEA (Fig. 3a).
Mechanical stress in RPE cells induces the expression of angiogenic and EMT factors and promotes in vitro angiogenesis
qRT-PCR results showed substantial changes in the expression of major angiogenic and EMT mRNA (Fig. 3q, r). Three hours after applying mechanical stress, the expression of angiogenic factors, VEGF121, HIF-1α, ANG2 and IL-8, increased significantly (p = 0.018, 0.007, 0.021, 0.018, respectively). The expression of pigment epithelium-derived factor (PEDF), an anti-angiogenic gene, was also increased (p = 0.044). We observed a significant increase in the expression of the EMT promoter, TNF-α (p = 0.005). After six hours, the expression of two major VEGF isoforms, VEGF121 and VEGF165, and a stimulator of VEGF expression, HIF-1α, increased (p = 0.011, 0.016, 0.011, respectively). Moreover, the expression of ANG2, IL-6 and IL-8, PEDF and TNF-α also increased significantly (p = 0.017, 0.011, 0.013, 0.013, 0.016, respectively; Fig. 3q). While FGF2 gene expression in mechanically stressed RPE cells decreased significantly after six hours (p = 0.017), there was no significant change in apical and basal FGF2 protein expression (p = 0.52, 0.49, respectively; Fig. 3q, s, t).
We also observed a significant increase in the following EMT markers, CDH2 (p(3 hours) = 0.044, p(6 hours) = 0.024), VIM (p(3 hours) = 0.094, p(6 hours) = 0.017), and FN1 (p(3 hours) = 0.044, p(6 hours) = 0.017), and a decrease in CDH1 (p(3 hours) = 0.044, p(6 hours) = 0.051) and an RPE-specific gene, RPE65 ( p(3 hours) = 0.03, p(6 hours) = 0.00; Fig. 3r). ELISA results showed a significant increase in the expression of IL-6, apically (p = 0.014 respectively), and VEGF, ANG2, IL-6 and IL-8, basolaterally (p = 0.042, 0.008, 0.000, 0.019 respectively; Fig. 3s, t).
qRT-PCR results showed a concurrent increase in pro-angiogenic gene expression, as mentioned above, as well as the anti-angiogenic factor, PEDF (Fig. 3q). To determine whether the increase of pro-angiogenic factors overrides the anti-angiogenic activity of PEDF, an in vitro angiogenesis assay was performed. Used media from RPE cultures activated endothelial tube formation, while limited angiogenic activity was observed in HUVECs cultured in fresh RPE media (Fig. 4). Exposure to apical and basal conditioned media from mechanically stressed RPE cells resulted in a significant ~ 2- and 1.4-fold increase in endothelial tube length (p = 0.001, 0.017, respectively) and ~ 3.6- and 2-fold increase in node number respectively (p = 0.003, 0.018, respectively; Fig. 4f, g).