Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado, United States of America.
PLoS One. 2012;7(7):e39969. doi: 10.1371/journal.pone.0039969. Epub 2012 Jul 13.
Fibroblasts residing in connective tissues throughout the body are responsible for extracellular matrix (ECM) homeostasis and repair. In response to tissue damage, they activate to become myofibroblasts, which have organized contractile cytoskeletons and produce a myriad of proteins for ECM remodeling. However, persistence of myofibroblasts can lead to fibrosis with excessive collagen deposition and tissue stiffening. Thus, understanding which signals regulate de-activation of myofibroblasts during normal tissue repair is critical. Substrate modulus has recently been shown to regulate fibrogenic properties, proliferation and apoptosis of fibroblasts isolated from different organs. However, few studies track the cellular responses of fibroblasts to dynamic changes in the microenvironmental modulus. Here, we utilized a light-responsive hydrogel system to probe the fate of valvular myofibroblasts when the Young's modulus of the substrate was reduced from ~32 kPa, mimicking pre-calcified diseased tissue, to ~7 kPa, mimicking healthy cardiac valve fibrosa. After softening the substrata, valvular myofibroblasts de-activated with decreases in α-smooth muscle actin (α-SMA) stress fibers and proliferation, indicating a dormant fibroblast state. Gene signatures of myofibroblasts (including α-SMA and connective tissue growth factor (CTGF)) were significantly down-regulated to fibroblast levels within 6 hours of in situ substrate elasticity reduction while a general fibroblast gene vimentin was not changed. Additionally, the de-activated fibroblasts were in a reversible state and could be re-activated to enter cell cycle by growth stimulation and to express fibrogenic genes, such as CTGF, collagen 1A1 and fibronectin 1, in response to TGF-β1. Our data suggest that lowering substrate modulus can serve as a cue to down-regulate the valvular myofibroblast phenotype resulting in a predominantly quiescent fibroblast population. These results provide insight in designing hydrogel substrates with physiologically relevant stiffness to dynamically redirect cell fate in vitro.
分布于全身结缔组织中的成纤维细胞负责细胞外基质（ECM）的稳态和修复。在组织损伤时，它们会激活成为肌成纤维细胞，后者具有组织化的收缩细胞骨架，并产生大量的 ECM 重塑蛋白。然而，肌成纤维细胞的持续存在可能导致纤维化，表现为胶原过度沉积和组织僵硬。因此，了解哪些信号调节正常组织修复过程中肌成纤维细胞的失活至关重要。最近的研究表明，基质模量调节源自不同器官的成纤维细胞的纤维生成特性、增殖和凋亡。然而，很少有研究追踪成纤维细胞对微环境模量动态变化的细胞反应。在这里，我们利用光响应水凝胶系统来研究瓣膜肌成纤维细胞的命运，即在将基底的杨氏模量从约 32 kPa（模拟预钙化病变组织）降低至约 7 kPa（模拟健康的心脏瓣膜纤维层）时的变化。在基底变软后，α-平滑肌肌动蛋白（α-SMA）应力纤维和增殖减少，表明肌成纤维细胞失活，进入休眠状态。在原位基底弹性降低 6 小时内，肌成纤维细胞的基因特征（包括α-SMA 和结缔组织生长因子（CTGF））显著下调至成纤维细胞水平，而一般的成纤维细胞基因波形蛋白（vimentin）没有变化。此外，失活的成纤维细胞处于可逆状态，可以通过生长刺激重新进入细胞周期，并在受到 TGF-β1 刺激时表达纤维生成基因，如 CTGF、胶原 1A1 和纤连蛋白 1。我们的数据表明，降低基底模量可以作为下调瓣膜肌成纤维细胞表型的信号，导致以静止为主的成纤维细胞群体。这些结果为设计具有生理相关弹性的水凝胶基底提供了思路，以便在体外动态改变细胞命运。
