Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Shanghai Key Laboratory of Fundus Disease, Shanghai, China.
Invest Ophthalmol Vis Sci. 2018 Jul 2;59(8):3619-3629. doi: 10.1167/iovs.17-23580.
Biomechanical properties changes and α-smooth muscle actin (α-SMA) overexpression are involved in myopia scleral remodeling. However, interactions between altered tissue biomechanics and cellular signaling that sustain scleral remodeling have not been well defined. We determine the mechanisms of mechanotransduction in the regulation of α-SMA expression during myopia scleral remodeling.
Guinea pigs were used to establish a form-deprivation myopia (FDM) model. Protein profiles in myopic sclera were examined using tandem mass spectrometry. Ras homolog gene family member A (RhoA) and α-SMA expressions were confirmed using quantitative (q) RT-PCR and Western blotting. Scleral fibroblasts were cultured and subjected to 4% cyclic strain. Levels of RhoA, rho-associated protein kinase-2 (ROCK2), myocardin-related transcription factor-A (MRTF-A), serum response factor (SRF), and α-SMA were determined by qRT-PCR and Western blotting in groups with or without the RhoA siRNA or ROCK inhibitor Y27632. MRTF-A and α-SMA were evaluated by confocal immunofluorescent microscopy and myofibroblasts were enumerated using flow cytometry.
mRNA and protein levels of RhoA and α-SMA were significantly increased in the FDM eyes after 4 weeks of form-deprivation treatment. The 4% static strain increased expressions of RhoA, ROCK2, MRTF-A, SRF, and α-SMA as well as nuclear translocalization of MRTF-A in scleral fibroblasts compared to those without strain stimulation. Additionally, the percentage of myofibroblasts increased after strain stimulation. Conversely, inhibition of RhoA or ROCK2 reversed the strain-induced α-SMA expression and myofibroblast ratio.
Mechanical strain activated RhoA signaling and scleral myofibroblast differentiation. Strain also mediated myofibroblast differentiation via the RhoA/ROCK2-MRTF-A/SRF pathway. These findings provided evidence for a mechanical strain-induced RhoA/ROCK2 pathway that may contribute to myopia scleral remodeling.
生物力学特性的改变和α-平滑肌肌动蛋白（α-SMA）的过表达与近视巩膜重塑有关。然而，改变的组织生物力学与维持巩膜重塑的细胞信号之间的相互作用尚未得到很好的定义。我们确定了机械转导在近视巩膜重塑过程中调节α-SMA 表达的机制。
使用豚鼠建立形觉剥夺性近视（FDM）模型。使用串联质谱法检测近视巩膜的蛋白质谱。使用定量（q）RT-PCR 和 Western blotting 验证 Ras 同源基因家族成员 A（RhoA）和α-SMA 的表达。培养巩膜成纤维细胞并施加 4%周期性应变。在存在或不存在 RhoA siRNA 或 ROCK 抑制剂 Y27632 的情况下，通过 qRT-PCR 和 Western blotting 测定各组中 RhoA、rho 相关蛋白激酶-2（ROCK2）、心肌营养素相关转录因子-A（MRTF-A）、血清反应因子（SRF）和α-SMA 的水平。通过共聚焦免疫荧光显微镜评估 MRTF-A 和α-SMA，并通过流式细胞术计数肌成纤维细胞。
在 4 周的形觉剥夺治疗后，FDM 眼中 RhoA 和α-SMA 的 mRNA 和蛋白水平均显著增加。与无应变刺激相比，4%的静态应变增加了巩膜成纤维细胞中 RhoA、ROCK2、MRTF-A、SRF 和α-SMA 的表达以及 MRTF-A 的核转位。此外，应变刺激后肌成纤维细胞的比例增加。相反，抑制 RhoA 或 ROCK2 逆转了应变诱导的α-SMA 表达和肌成纤维细胞比值。
机械应变激活了 RhoA 信号通路并诱导巩膜肌成纤维细胞分化。应变还通过 RhoA/ROCK2-MRTF-A/SRF 通路介导肌成纤维细胞分化。这些发现为机械应变诱导的 RhoA/ROCK2 通路提供了证据，该通路可能与近视巩膜重塑有关。
