Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI, 48105, USA.
KCL Centre for Cell and Gene Therapy, London, WC2R 2LS, England, UK.
Sci Rep. 2022 Jul 26;12(1):12694. doi: 10.1038/s41598-022-16701-6.
The retinal pigment epithelium (RPE) is a polarized monolayer that secretes growth factors and cytokines towards the retina apically and the choroid basolaterally. Numerous RPE secreted proteins have been linked to the pathogenesis of age-related macular degeneration (AMD). The purpose of this study was to determine the differential apical and basolateral secretome of RPE cells, and the effects of oxidative stress on directional secretion of proteins linked to AMD and angiogenesis. Tandem mass tag spectrometry was used to profile proteins in human iPSC-RPE apical and basolateral conditioned media. Changes in secretion after oxidative stress induced by HO or tert-butyl hydroperoxide (tBH) were investigated by ELISA and western analysis. Out of 926 differentially secreted proteins, 890 (96%) were more apical. Oxidative stress altered the secretion of multiple factors implicated in AMD and neovascularization and promoted a pro-angiogenic microenvironment by increasing the secretion of pro-angiogenic molecules (VEGF, PTN, and CRYAB) and decreasing the secretion of anti-angiogenic molecules (PEDF and CFH). Apical secretion was impacted more than basolateral for PEDF, CRYAB and CFH, while basolateral secretion was impacted more for VEGF, which may have implications for choroidal neovascularization. This study lays a foundation for investigations of dysfunctional RPE polarized protein secretion in AMD and other chorioretinal degenerative disorders.
视网膜色素上皮 (RPE) 是一种极化的单层细胞，它向视网膜顶部分泌生长因子和细胞因子，向脉络膜基底外侧分泌。许多 RPE 分泌的蛋白质与年龄相关性黄斑变性 (AMD) 的发病机制有关。本研究旨在确定 RPE 细胞顶部分泌和基底外侧分泌的差异，并研究氧化应激对与 AMD 和血管生成相关的蛋白质定向分泌的影响。串联质量标签质谱用于分析人诱导多能干细胞-RPE 顶部分泌和基底外侧条件培养基中的蛋白质。通过 ELISA 和 Western 分析研究了 HO 或叔丁基过氧化物 (tBH) 诱导的氧化应激后分泌的变化。在 926 种差异分泌的蛋白质中，有 890 种（96%）更偏向于顶部分泌。氧化应激改变了多种与 AMD 和血管新生有关的因子的分泌，并通过增加促血管生成分子（VEGF、PTN 和 CRYAB）的分泌和减少抗血管生成分子（PEDF 和 CFH）的分泌，促进了促血管生成的微环境。PEDF、CRYAB 和 CFH 的顶部分泌比基底外侧分泌受影响更大，而 VEGF 的基底外侧分泌受影响更大，这可能对脉络膜新生血管化有影响。本研究为研究 AMD 和其他脉络膜视网膜退行性疾病中功能失调的 RPE 极化蛋白分泌奠定了基础。
