After surgery or traumatic injury, corneal wound healing can cause a scarring response that stiffens the tissue and impairs ocular function. This fibrosis is caused in part by the activation of corneal keratocytes from a native mechanically quiescent state to an activated myofibroblastic state. This transformation is tied to signaling downstream of transforming growth factor-β1 (TGF-β1). Here, to better understand how biochemical and biophysical cues interact to regulate keratocyte activation and contractility, we cultured primary rabbit corneal keratocytes on flexible substrata of varying stiffness in the presence (or absence) of TGF-β1. Time-lapse fluorescence microscopy was used to assess changes in keratocyte morphology, as well as to quantify the dynamic traction stresses exerted by cells under different experimental conditions. In other experiments, keratocytes were fixed after 5 days of culture and stained for markers of both contractility and myofibroblastic activation. Treatment with TGF-β1 elicited distinct phenotypes on substrata of different stiffnesses. Cells on soft (1 kPa) gels formed fewer stress fibers and retained a more dendritic morphology, indicative of a quiescent keratocyte phenotype. Keratocytes cultured on stiff (10 kPa) gels or collagen-coated glass coverslips, however, had broad morphologies, formed abundant stress fibers, exhibited greater levels of α-smooth muscle actin (α-SMA) expression, and exerted larger traction forces. Confocal images of phospho-myosin light chain (pMLC) immunofluorescence, moreover, revealed stiffness-dependent differences in the subcellular distribution of actomyosin contractility, with pMLC localized at the tips of thin cellular processes in mechanically quiescent cells. Importantly, keratocytes cultured in the absence of TGF-β1 showed no stiffness-dependent differences in α-SMA immunofluorescence, suggesting that a stiff microenvironment alone is insufficient to induce myofibroblastic activation. Taken together, these data suggest that changes in ECM stiffness can modulate the morphology, cytoskeletal organization, and subcellular pattern of force generation in corneal keratocytes treated with TGF-β1.

中文翻译：

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ECM 刚度控制响应 TGF-β1 的角膜角膜细胞的激活和收缩性

手术或外伤后，角膜伤口愈合会引起疤痕反应，使组织变硬并损害眼部功能。这种纤维化部分是由角膜角质细胞从天然机械静止状态激活到激活的肌成纤维细胞状态引起的。这种转化与转化生长因子-β1 (TGF-β1) 的下游信号传导有关。在这里，为了更好地了解生化和生物物理线索如何相互作用以调节角膜细胞活化和收缩性，我们在存在（或不存在）TGF-β1 的情况下在不同硬度的柔性基质上培养了原代兔角膜角膜细胞。延时荧光显微镜用于评估角膜细胞形态的变化，以及量化细胞在不同实验条件下施加的动态牵引应力。在其他实验中，角膜细胞在培养 5 天后被固定，并对收缩性和肌成纤维细胞活化的标记进行染色。用 TGF-β1 处理在不同硬度的基质上引起不同的表型。软 (1 kPa) 凝胶上的细胞形成较少的应力纤维并保留更多的树突形态，表明是静止的角膜细胞表型。然而，在硬 (10 kPa) 凝胶或胶原涂层玻璃盖玻片上培养的角质细胞具有广泛的形态，形成丰富的应力纤维，表现出更高水平的 α-平滑肌肌动蛋白 (α-SMA) 表达，并施加更大的牵引力。此外，磷酸肌球蛋白轻链 (pMLC) 免疫荧光的共聚焦图像揭示了肌动球蛋白收缩性亚细胞分布的刚度依赖性差异，pMLC 位于机械静止细胞中薄细胞突起的尖端。重要的是，在没有 TGF-β1 的情况下培养的角膜细胞在 α-SMA 免疫荧光中没有显示出硬度依赖性差异，这表明单独的僵硬微环境不足以诱导肌成纤维细胞活化。总之，这些数据表明 ECM 刚度的变化可以调节用 TGF-β1 处理的角膜角膜细胞的形态、细胞骨架组织和力产生的亚细胞模式。