Department of Chemical Engineering, University of Virginia, USA.
Department of Biomedical Engineering, University of Virginia, USA.
Biomater Sci. 2023 Apr 11;11(8):2886-2897. doi: 10.1039/d2bm02058k.
Cellular mechanotransduction plays a central role in fibroblast activation during fibrotic disease progression, leading to increased tissue stiffness and reduced organ function. While the role of epigenetics in disease mechanotransduction has begun to be appreciated, little is known about how substrate mechanics, particularly the timing of mechanical inputs, regulate epigenetic changes such as DNA methylation and chromatin reorganization during fibroblast activation. In this work, we engineered a hyaluronic acid hydrogel platform with independently tunable stiffness and viscoelasticity to model normal (storage modulus, ' ∼ 0.5 kPa, loss modulus, '' ∼ 0.05 kPa) to increasingly fibrotic (' ∼ 2.5 and 8 kPa, '' ∼ 0.05 kPa) lung mechanics. Human lung fibroblasts exhibited increased spreading and nuclear localization of myocardin-related transcription factor-A (MRTF-A) with increasing substrate stiffness within 1 day, with these trends holding steady for longer cultures. However, fibroblasts displayed time-dependent changes in global DNA methylation and chromatin organization. Fibroblasts initially displayed increased DNA methylation and chromatin decondensation on stiffer hydrogels, but both of these measures decreased with longer culture times. To investigate how culture time affected the responsiveness of fibroblast nuclear remodeling to mechanical signals, we engineered hydrogels amenable to secondary crosslinking, enabling a transition from a compliant substrate mimicking normal tissue to a stiffer substrate resembling fibrotic tissue. When stiffening was initiated after only 1 day of culture, fibroblasts rapidly responded and displayed increased DNA methylation and chromatin decondensation, similar to fibroblasts on static stiffer hydrogels. Conversely, when fibroblasts experienced later stiffening at day 7, they showed no changes in DNA methylation and chromatin condensation, suggesting the induction of a persistent fibroblast phenotype. These results highlight the time-dependent nuclear changes associated with fibroblast activation in response to dynamic mechanical perturbations and may provide mechanisms to target for controlling fibroblast activation.
细胞力学转导在纤维化疾病进展过程中对成纤维细胞的激活起着核心作用，导致组织硬度增加和器官功能降低。虽然表观遗传学在疾病力学转导中的作用已经开始被认识，但对于基质力学（尤其是机械输入的时间）如何调节成纤维细胞激活过程中的表观遗传变化，如 DNA 甲基化和染色质重构，知之甚少。在这项工作中，我们设计了一种具有独立可调硬度和粘弹性的透明质酸水凝胶平台，以模拟正常（储能模量，“ ∼ 0.5 kPa，损耗模量，” ∼ 0.05 kPa）到越来越纤维化（“ ∼ 2.5 和 8 kPa，“ ∼ 0.05 kPa）的肺力学。人肺成纤维细胞在 1 天内表现出随着基质刚度的增加而增加的扩散和心肌相关转录因子-A（MRTF-A）的核定位，这些趋势在更长的培养时间内保持稳定。然而，成纤维细胞的全基因组 DNA 甲基化和染色质组织呈现时间依赖性变化。成纤维细胞最初在较硬的水凝胶上显示出增加的 DNA 甲基化和染色质去凝聚，但这两种措施随着培养时间的延长而降低。为了研究培养时间如何影响成纤维细胞核重塑对机械信号的反应性，我们设计了便于二次交联的水凝胶，使它从模拟正常组织的柔顺基质转变为模拟纤维化组织的刚性基质。当在培养 1 天后仅开始变硬时，成纤维细胞迅速响应并显示出增加的 DNA 甲基化和染色质去凝聚，类似于在静态刚性水凝胶上的成纤维细胞。相反，当成纤维细胞在第 7 天经历较晚的变硬时，它们的 DNA 甲基化和染色质凝聚没有变化，这表明诱导了持久的成纤维细胞表型。这些结果突出了成纤维细胞激活过程中与动态力学扰动相关的核变化的时间依赖性，并可能为控制成纤维细胞激活提供靶向机制。
