Department of Ophthalmology, USA; Center for Nanomedicine, USA.
Department of Ophthalmology, USA; Glaucoma Center of Excellence, The Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.
Exp Eye Res. 2024 Jul;244:109917. doi: 10.1016/j.exer.2024.109917. Epub 2024 Apr 30.
In glaucoma, scleral fibroblasts are exposed to IOP-associated mechanical strain and elevated TGFβ levels. These stimuli, in turn, lead to scleral remodeling. Here, we examine the scleral fibroblast migratory and transcriptional response to these stimuli to better understand mechanisms of glaucomatous scleral remodeling. Human peripapillary scleral (PPS) fibroblasts were cultured on parallel grooves, treated with TGFβ (2 ng/ml) in the presence of vehicle or TGFβ signaling inhibitors, and exposed to uniaxial strain (1 Hz, 5%, 12-24 h). Axis of cellular orientation was determined at baseline, immediately following strain, and 24 h after strain cessation with 0° being completely aligned with grooves and 90° being perpendicular. Fibroblasts migration in-line and across grooves was assessed using a scratch assay. Transcriptional profiling of TGFβ-treated fibroblasts with or without strain was performed by RT-qPCR and pERK, pSMAD2, and pSMAD3 levels were measured by immunoblot. Pre-strain alignment of TGFβ-treated cells with grooves (6.2 ± 1.5°) was reduced after strain (21.7 ± 5.3°, p < 0.0001) and restored 24 h after strain cessation (9.5 ± 2.6°). ERK, FAK, and ALK5 inhibition prevented this reduction; however, ROCK, YAP, or SMAD3 inhibition did not. TGFβ-induced myofibroblast markers were reduced by strain (αSMA, POSTN, ASPN, MLCK1). While TGFβ-induced phosphorylation of ERK and SMAD2 was unaffected by cyclic strain, SMAD3 phosphorylation was reduced (p = 0.0004). Wound healing across grooves was enhanced by ROCK and SMAD3 inhibition but not ERK or ALK5 inhibition. These results provide insight into the mechanisms by which mechanical strain alters the cellular response to TGFβ and the potential signaling pathways that underlie scleral remodeling.
在青光眼患者中，巩膜成纤维细胞暴露于与眼压相关的机械应变和升高的 TGFβ 水平下。这些刺激反过来导致巩膜重塑。在这里，我们研究了巩膜成纤维细胞对这些刺激的迁移和转录反应，以更好地理解青光眼巩膜重塑的机制。我们培养人眼周围巩膜（PPS）成纤维细胞在平行的凹槽上，用 TGFβ（2ng/ml）处理，同时用 TGFβ 信号抑制剂或载体处理，然后施加单轴应变（1Hz，5%，12-24 小时）。在基线、应变后立即和应变停止后 24 小时，通过将细胞的取向轴与凹槽对齐，确定 0°表示完全与凹槽对齐，90°表示垂直于凹槽。通过划痕实验评估成纤维细胞在凹槽内和横跨凹槽的迁移。用或不用应变的 TGFβ 处理的成纤维细胞进行转录谱分析，通过 RT-qPCR 进行，通过免疫印迹测量 pERK、pSMAD2 和 pSMAD3 水平。在应变前，TGFβ 处理的细胞与凹槽对齐（6.2±1.5°），在应变后减少（21.7±5.3°，p<0.0001），并在应变停止后 24 小时恢复（9.5±2.6°）。ERK、FAK 和 ALK5 抑制阻止了这种减少；然而，ROCK、YAP 或 SMAD3 抑制没有。应变降低了 TGFβ 诱导的肌成纤维细胞标志物（αSMA、POSTN、ASPN、MLCK1）。虽然循环应变对 TGFβ 诱导的 ERK 和 SMAD2 磷酸化没有影响，但 SMAD3 磷酸化减少（p=0.0004）。ROCK 和 SMAD3 抑制增强了跨越凹槽的伤口愈合，但 ERK 或 ALK5 抑制没有。这些结果提供了对机械应变改变细胞对 TGFβ 反应的机制的深入了解，以及潜在的信号通路，这些通路是巩膜重塑的基础。
