Doheny Eye Institute and Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
Doheny Eye Institute and Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA; Eye Hospital of China Academy of Chinese Medical Sciences, Beijing, China.
Exp Eye Res. 2019 Oct;187:107774. doi: 10.1016/j.exer.2019.107774. Epub 2019 Aug 23.
Steroid-induced ocular hypertension can be seen even after trabecular meshwork (TM) bypass/ablation. Thus, the purpose was to investigate steroid-response in cells distal to the TM by using primary scleral fibroblasts. Primary scleral cell cultures were generated using mid-depth scleral wedges from human donor corneo-scleral rims (n = 5) after corneal transplantation. Cells were treated with dexamethasone (DEX; 100 nM) and compared to media (MED)/vehicle (DMSO) controls. Cell size, shape, and migration were studied using the IncuCyte Live-Cell Analysis System. Cytoskeleton was compared using Alexa Fluor-568 Phalloidin and senescence tested by evaluating beta-galactosidase. Western blot comparison was performed for α-SMA, FKBP-51, fibronectin, phospho-myosin light chain, and myocilin. Scleral fibroblasts upregulated FKBP-51 in response to DEX indicating the existence of steroid-responsive pathways. Compared to controls, DEX-treated cells proliferated slower (50%; p < 0.01-0.02), grew larger (1.3-fold; p < 0.001), and migrated less (p = 0.01-0.006). Alexa Fluor 568 Phalloidin actin stress fiber labeling was more diffuse in DEX-treated cells (p = 0.001-0.004). DEX-treated cells showed more senescence compared to controls (~1.7-fold; p = 0.01-0.02). However, DEX-treated cells did not show increased cross-linked actin network formation or elevated myocilin/fibronectin/α-SMA/phospho-myosin light chain protein expression. For all parameters, MED- and DMSO-treated control cells were not significantly different. Primary scleral fibroblasts, grown from tissue collected immediately distal to the TM, demonstrated scleral-response behaviors that were similar to, but not identical with, classic TM steroid-response. Further study is needed to understand how these scleral cellular alterations may contribute to steroid-response IOP elevation after TM bypass/ablation surgery.
即使在小梁网（TM）旁路/消融术后，也可能会出现类固醇诱导的眼压升高。因此，本研究的目的是通过使用原发性巩膜成纤维细胞来研究 TM 远端的类固醇反应。使用角膜移植后从人供体角膜巩膜缘获得的中深度巩膜楔形物（n=5）生成原发性巩膜细胞培养物。用地塞米松（DEX；100 nM）处理细胞，并与培养基（MED）/载体（DMSO）对照进行比较。使用 IncuCyte 活细胞分析系统研究细胞大小、形状和迁移。使用 Alexa Fluor-568 鬼笔环肽比较细胞骨架，并通过评估β-半乳糖苷酶来测试衰老。进行 Western blot 比较以评估α-SMA、FKBP-51、纤连蛋白、磷酸肌球蛋白轻链和肌球蛋白。巩膜成纤维细胞对 DEX 的反应上调 FKBP-51，表明存在类固醇反应途径。与对照组相比，DEX 处理的细胞增殖较慢（50%；p<0.01-0.02），生长较大（1.3 倍；p<0.001），迁移较少（p=0.01-0.006）。DEX 处理的细胞中的 Alexa Fluor 568 鬼笔环肽肌动蛋白应力纤维标记物的弥散程度更高（p=0.001-0.004）。与对照组相比，DEX 处理的细胞显示出更多的衰老（~1.7 倍；p=0.01-0.02）。然而，DEX 处理的细胞没有显示出交联肌动蛋白网络形成增加或肌球蛋白/纤连蛋白/α-SMA/磷酸肌球蛋白轻链蛋白表达升高。对于所有参数，MED 和 DMSO 处理的对照细胞均无显著差异。从 TM 直接远端收集的组织中培养的原发性巩膜成纤维细胞表现出与经典 TM 类固醇反应相似但不完全相同的巩膜反应行为。需要进一步研究以了解这些巩膜细胞变化如何导致 TM 旁路/消融术后类固醇反应性眼压升高。
