The impact of early RPE cell junction loss on VEGF, Ang-2, and TIMP secretion in vitro.

Purpose: The retinal pigment epithelium (RPE) is an important tissue for maintaining a healthy retina. Retinal pigment epithelial cells help regulate nutrient transport to photoreceptors and are heavily pigmented to prevent light scattering. These cells also have junction proteins to form monolayers. Monolayers are key players in pathologies such as age-related macular degeneration (AMD), a leading cause of vision loss in older adults. During AMD, RPE cell detachment can occur, resulting in a loss of junctions. Losing junctions can increase the expression of pro-angiogenic vascular endothelial growth factor (VEGF). This overexpression can cause abnormal blood vessel growth or angiogenesis in the retina. Age-related macular degeneration treatments target VEGF to slow angiogenesis progression. However, other proteins, such as angiopoietin-2 (Ang-2) and the tissue inhibitor of metalloproteinase-1 (TIMP-1), may also play important roles, making them potential targets for treatment. Controlling RPE junction formation will help elucidate the relationship between RPE cell detachment and additional angiogenic factor secretion, lead to more therapeutics, and increase the efficacy of current treatments. Methods: Micropatterning was used to control the spatial arrangement of primary porcine RPE cells using polydimethylsiloxane (PDMS) stencils. Patterns were formed into PDMS stencils to mimic 10%, 25%, and 50% overall detachment of the RPE monolayer. Zonula-occludens-1 (ZO-1), Ang-2, and VEGF were visualized using immunocytochemical (ICC) staining. An enzyme-linked immunosorbent assay (ELISA) was used to quantify extracellular Ang-2, VEGF, TIMP-1, and TIMP-2 levels. A rod outer segment (OS) phagocytosis assay was performed to determine how RPE junction loss directly affects photoreceptor support. Results: The growth of primary porcine RPE cells was successfully controlled using stencils. Morphological changes and a decrease in pigmentation were observed, showing a decline in barrier and light absorption functions as degeneration increased. One day after stencil removal, junction proteins were delocalized, and angiogenic factor secretions were correlated with increased levels of detachment. Secretion levels of Ang-2 and TIMP-1 were significantly increased, whereas VEGF and TIMP-2 concentrations were not as affected by varying levels of detachment. OS phagocytosis appeared lower in RPE cells when ZO-1 was affected. Conclusions: These results suggest a correlation between loss of junctions, abnormal angiogenic protein secretion, and reduced OS phagocytosis. Furthermore, Ang-2 and TIMP-1 proteins might be beneficial targets for AMD treatments, and their roles in retinal diseases deserve further investigation.

Utilizing Recombinant Spider Silk Proteins To Develop a Synthetic Bruch's Membrane for Modeling the Retinal Pigment Epithelium.

Spider silks are intriguing biomaterials that have a high potential as innovative biomedical processes and devices. The intent of this study was to evaluate the capacity of recombinant spider silk proteins (rSSps) as a synthetic Bruch's membrane. Nonporous silk membranes were prepared with comparable thicknesses (<10 µm) to that of native Bruch's membrane. Biomechanical characterization was performed prior to seeding cells. The ability of RPE cells (ARPE-19) to attach and grow on the membranes was then evaluated with bright-field and electron microscopy, intracellular DNA quantification, and immunocytochemical staining (ZO-1 and F-actin). Controls were cultured on permeable Transwell support membranes and characterized with the same methods. A size-dependent permeability assay, using FITC-dextran, was used to determine cell-membrane barrier function. Compared to Transwell controls, RPE cells cultured on rSSps membranes developed more native-like "cobblestone" morphologies, exhibited higher intracellular DNA content, and expressed key organizational proteins more consistently. Comparisons of the membranes to native structures revealed that the silk membranes exhibited equivalent thicknesses, biomechanical properties, and barrier functions. These findings support the use of recombinant spider silk proteins to model Bruch's membrane and develop more biomimetic retinal models.