CALL FOR PAPERS Bioengineering the Lung: Molecules, Materials, Matrix, Morphology, and Mechanics Improved throughput traction microscopy reveals pivotal role for matrix stiffness in fibroblast contractility and TGF- (cid:1) responsiveness
CALL FOR PAPERS Bioengineering the Lung: Molecules, Materials, Matrix, Morphology, and Mechanics Improved throughput traction microscopy reveals pivotal role for matrix stiffness in fibroblast contractility and TGF- (cid:1) responsiveness
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改进的吞吐量牵引显微镜揭示了基质刚度在成纤维细胞收缩性和 TGF-(cid:1) 反应性中的关键作用。生理成纤维细胞功能，例如基质重塑和潜在转化生长因子- (cid:1) 1 (TGF- (cid:1) 1) 的激活与肌成纤维细胞表型的表达相关，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对成纤维细胞力产生能力的研究受到低通量和耗时的程序的阻碍。在这项研究中，我们在细节水平上改进了聚丙烯酰胺水凝胶上高通量牵引力测量的方法，使用凝胶表面结合的荧光珠以允许自动聚焦和自动位移图，以及用荧光标记转导成纤维细胞以简化细胞边界识别。这些进步共同显着提高了牵引显微镜的通量，并使我们能够有效地计算肺成纤维细胞对跨越正常和纤维化肺组织中存在的硬度范围的基底施加的力。我们的结果表明，随着细胞外基质硬度的变化，肺成纤维细胞会显着改变它们传递到细胞外基质的力，基质上产生的力非常低，与正常肺组织一样顺应。此外，外源性TGF-(cid:1) 1选择性地增强对僵硬基质的牵引力，模仿纤维化肺，但不增强对生理僵硬基质的牵引力，尽管Smad2/3激活发生了相同的变化。总而言之，这些结果证明了基质机械特性在调节基线和 TGF-(cid:1)1 刺激的肺成纤维细胞收缩中的关键作用，并表明僵硬的纤维化肺组织可能通过收缩驱动事件促进肌成纤维细胞活化，而正常的肺组织顺应性可能会防止成纤维细胞活化的这种反馈放大。简而言之，通过使用 Otsu 方法应用全局图像阈值，从荧光图像中提取细胞核和投影细胞区域 (47)。这些区域进一步用于提取和整合细胞核和细胞质磷酸化 Smad2/3 荧光。最后，对于每个图像，每单位面积的核磷酸化 Smad2/3 荧光的积分强度被无量纲化为每单位面积的细胞质磷酸化 Smad2/3 荧光的积分强度的比率。统计分析。线性回归分析在 Excel (Microsoft) 中完成。在 Stata 统计软件 (StataCorp LP) 中进行单向方差分析，然后进行 Tukey 检验或双尾 t 检验。
Improved throughput traction microscopy reveals pivotal role for matrix stiffness in fibroblast contractility and TGF- (cid:1) responsiveness. Physiol fibroblast functions such as matrix remodeling and activation of latent transforming growth factor- (cid:1) 1 (TGF- (cid:1) 1) 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 sub-stantially 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- (cid:1) 1 selectively accen-tuates 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- (cid:1) 1-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. Briefly, nuclei and projected cell areas were extracted from the fluorescent images by applying a global image threshold using Otsu’s method (47). Those areas were further used to extract and integrate nuclear and cytoplasmic phospho-Smad2/3 fluorescence. Finally, for each image, integrated intensity of nuclear phospho-Smad2/3 fluorescence per unit area was nondimensionalized as a ratio of the integrated intensity of cytoplasmic phospho-Smad2/3 fluorescence per unit area. Statistical analysis. Linear regression analysis was done in Excel (Microsoft). One-way ANOVA followed by Tukey’s test or two-tailed t -test were done in Stata statistical software (StataCorp LP).