Molecular and Integrative Physiological Sciences, Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Am J Physiol Lung Cell Mol Physiol. 2012 Aug 1;303(3):L169-80. doi: 10.1152/ajplung.00108.2012. Epub 2012 Jun 1.
Lung fibroblast functions such as matrix remodeling and activation of latent transforming growth factor-β1 (TGF-β1) are associated with expression of the myofibroblast phenotype and are directly linked to fibroblast capacity to generate force and deform the extracellular matrix. However, the study of fibroblast force-generating capacities through methods such as traction force microscopy is hindered by low throughput and time-consuming procedures. In this study, we improved at the detail level methods for higher-throughput traction measurements on polyacrylamide hydrogels using gel-surface-bound fluorescent beads to permit autofocusing and automated displacement mapping, and transduction of fibroblasts with a fluorescent label to streamline cell boundary identification. Together these advances substantially improve the throughput of traction microscopy and allow us to efficiently compute the forces exerted by lung fibroblasts on substrates spanning the stiffness range present in normal and fibrotic lung tissue. Our results reveal that lung fibroblasts dramatically alter the forces they transmit to the extracellular matrix as its stiffness changes, with very low forces generated on matrices as compliant as normal lung tissue. Moreover, exogenous TGF-β1 selectively accentuates tractions on stiff matrices, mimicking fibrotic lung, but not on physiological stiffness matrices, despite equivalent changes in Smad2/3 activation. Taken together, these results demonstrate a pivotal role for matrix mechanical properties in regulating baseline and TGF-β1-stimulated contraction of lung fibroblasts and suggest that stiff fibrotic lung tissue may promote myofibroblast activation through contractility-driven events, whereas normal lung tissue compliance may protect against such feedback amplification of fibroblast activation.
肺成纤维细胞的功能，如基质重塑和潜伏转化生长因子-β1（TGF-β1）的激活，与肌成纤维细胞表型的表达有关，并且与成纤维细胞产生力和使细胞外基质变形的能力直接相关。然而，通过牵引力显微镜等方法研究成纤维细胞产生力的能力受到低通量和耗时的程序的限制。在这项研究中，我们通过使用表面结合荧光珠的聚丙烯酰胺水凝胶来提高高通量牵引力测量的细节水平，从而实现自动对焦和自动位移映射，并通过荧光标记转染成纤维细胞，以简化细胞边界识别，从而改进了方法。这些改进显著提高了牵引力显微镜的通量，并使我们能够有效地计算肺成纤维细胞在跨越正常和纤维化肺组织中存在的刚度范围内的基质上施加的力。我们的结果表明，肺成纤维细胞随着基质刚度的变化而显著改变它们向细胞外基质传递的力，在与正常肺组织一样柔软的基质上产生的力非常小。此外，外源性 TGF-β1 选择性地强调了在刚性基质上的牵引力，模拟了纤维化的肺，但不在生理刚度基质上，尽管 Smad2/3 激活发生了等效变化。总之，这些结果表明，基质力学性质在调节肺成纤维细胞的基础和 TGF-β1 刺激收缩中起着关键作用，并表明刚性纤维化肺组织可能通过收缩性驱动事件促进肌成纤维细胞的激活，而正常肺组织的顺应性可能会防止成纤维细胞激活的这种反馈放大。
