Department of Cellular Biology and Anatomy, Augusta University, Augusta, GA, United States.
College of Optometry, University of Houston, Houston, TX, United States.
Exp Eye Res. 2024 Nov;248:110069. doi: 10.1016/j.exer.2024.110069. Epub 2024 Sep 2.
Pathologic bidirectional interactions between the extracellular matrix (ECM) and cells within the human trabecular meshwork (hTM) contribute to ocular hypertension. An in vitro model is needed to study these cell-matrix interactions and their effect on outflow homeostasis. This study aimed to determine whether pathogenic ECM derived from dexamethasone (DEX)-treated hTM cultures induces clinically relevant glaucoma-like changes in healthy hTM cells at the transcriptional level. Corneoscleral rims from non-glaucoma donors were used to isolate primary hTM cells after validation according to the consensus recommendations for TM culture. Normal hTM cells (n = 5) were plated on a coverslip and treated with 100 nM DEX or ethanol for four weeks. These cultures were then decellularized, plated with primary hTM cells, and allowed to grow for another 72 h. RNA was extracted from these hTM cells for stranded total RNA-Seq. Sequencing libraries prepared using the Zymo-Seq RiboFree Total RNA library kit were pooled and sequenced using Illumina NovaSeq 6000. After quality control, sequence reads were aligned to the human genome build hg19. Differential expression (DE) analyses were performed using paired multi-factorial ANOVA. The expression of several DE genes associated with glaucoma (ANGPTL2, PDE7B, C22orf23, COL4A1, ADAM12, IFT122, SEMA6C) was validated using EvaGreen-based Droplet Digital PCR (ddPCR) assays. Gene ontology analyses of the DE genes were performed using the PANTHER and NDEx IQA databases, and functional analyses were performed with the DAVID Bioinformatics software. Using a cutoff of p-value <0.05 and fold change ≥2.0, our differential analysis identified 267 up- and 135 down-regulated genes in DEX-induced ECM-treated cells compared to the control. These differentially expressed genes were found to play a significant role in pathways such as cytokine and oxidative stress-induced inflammation, integrin signaling, matrix remodeling, and angiogenesis. These findings were further supported by previously performed proteomics studies using the same model. Using ddPCR, we validated the expression of seven genes associated with the risk of primary open-angle glaucoma. These results not only provide support for the pathogenic ECM model of steroid-induced glaucoma, but also demonstrate that the pathologic changes induced by this model are indeed found at the transcriptional level. These findings further demonstrate that matrix changes significantly influence cell expression profiles, which enable further understanding of the molecular mechanisms underlying glaucomatous changes in the TM. However, future studies with a larger and more diverse set of samples and longer time points are needed to confirm the utility of this model for mechanistic studies.
细胞外基质（ECM）与人类小梁网（hTM）内细胞之间的病理性双向相互作用导致眼内高压。需要体外模型来研究这些细胞-基质相互作用及其对流出物动态平衡的影响。本研究旨在确定源自地塞米松（DEX）处理的 hTM 培养物的致病 ECM 是否会在转录水平上诱导健康 hTM 细胞产生与临床相关的青光眼样变化。根据 TM 培养的共识建议，使用非青光眼供体的角膜缘来分离原代 hTM 细胞。将正常的 hTM 细胞（n=5）接种在盖玻片上，并用 100 nM DEX 或乙醇处理 4 周。然后将这些培养物去细胞化，接种原代 hTM 细胞，再培养 72 小时。从这些 hTM 细胞中提取 RNA 进行有向总 RNA-Seq。使用 Zymo-Seq RiboFree Total RNA 文库试剂盒制备测序文库，然后使用 Illumina NovaSeq 6000 进行测序。经过质量控制后，将序列读取与人类基因组 hg19 进行比对。使用配对多因素方差分析进行差异表达（DE）分析。使用 EvaGreen 基于的 Droplet Digital PCR（ddPCR）测定法验证与青光眼相关的几个 DE 基因（ANGPTL2、PDE7B、C22orf23、COL4A1、ADAM12、IFT122、SEMA6C）的表达。使用 PANTHER 和 NDEx IQA 数据库对 DE 基因进行基因本体分析，并使用 DAVID 生物信息学软件进行功能分析。使用 p 值<0.05 和倍数变化≥2.0 的截止值，我们的差异分析确定了与对照相比，DEX 诱导的 ECM 处理细胞中 267 个上调和 135 个下调基因。这些差异表达基因被发现参与了细胞因子和氧化应激诱导的炎症、整合素信号转导、基质重塑和血管生成等途径。这些发现得到了使用相同模型进行的先前蛋白质组学研究的支持。使用 ddPCR，我们验证了与原发性开角型青光眼风险相关的七个基因的表达。这些结果不仅为类固醇诱导性青光眼的致病 ECM 模型提供了支持，还表明该模型诱导的病理变化确实在转录水平上存在。这些发现进一步表明，基质变化显著影响细胞表达谱，这使我们能够进一步了解 TM 中青光眼变化的分子机制。然而，需要更多和更多样化的样本和更长时间点的未来研究来确认该模型在机制研究中的效用。
