Utah State University, Biological Engineering, Logan, UT.
Mol Vis. 2023 Jul 16;29:87-101. eCollection 2023.
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.
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.
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.
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.
视网膜色素上皮（RPE）是维持健康视网膜的重要组织。RPE 细胞有助于调节向光感受器的营养物质运输，并通过大量色素沉着来防止光散射。这些细胞还具有连接蛋白以形成单层。在诸如年龄相关性黄斑变性（AMD）等病理学中，单层是关键因素，AMD 是老年人视力丧失的主要原因。在 AMD 中，RPE 细胞可能会脱离，从而导致连接丢失。失去连接会增加促血管生成血管内皮生长因子（VEGF）的表达。这种过表达会导致视网膜中异常的血管生成或血管生成。针对 AMD 的治疗方法针对 VEGF 以减缓血管生成的进展。但是，其他蛋白质，例如血管生成素-2（Ang-2）和基质金属蛋白酶组织抑制剂-1（TIMP-1），也可能起重要作用，因此它们可能成为治疗的潜在靶标。控制 RPE 连接的形成将有助于阐明 RPE 细胞脱离与其他血管生成因子分泌之间的关系，从而导致更多的治疗方法，并提高当前治疗方法的功效。
使用聚二甲基硅氧烷（PDMS）模板通过微图案化来控制原代猪 RPE 细胞的空间排列。通过形成 PDMS 模板来模拟 RPE 单层的 10％，25％和 50％的总脱离。使用免疫细胞化学（ICC）染色来可视化封闭蛋白-1（ZO-1），Ang-2 和 VEGF。酶联免疫吸附测定（ELISA）用于定量细胞外 Ang-2，VEGF，TIMP-1 和 TIMP-2 水平。进行杆状光感受器外节（OS）吞噬作用测定以确定 RPE 连接丢失如何直接影响光感受器的支持。
使用模板成功控制了原代猪 RPE 细胞的生长。观察到形态变化和色素沉着减少，表明随着变性的增加，屏障和光吸收功能下降。模板去除后 1 天，连接蛋白发生了定位变化，并且随着脱离程度的增加，血管生成因子的分泌也随之增加。Ang-2 和 TIMP-1 的分泌水平显着增加，而 VEGF 和 TIMP-2 的浓度则不受脱离程度变化的影响。当 ZO-1 受到影响时，RPE 细胞中的 OS 吞噬作用似乎较低。
这些结果表明连接丢失，异常血管生成蛋白分泌和 OS 吞噬作用降低之间存在相关性。此外，Ang-2 和 TIMP-1 蛋白可能是 AMD 治疗的有益靶标，它们在视网膜疾病中的作用值得进一步研究。
