Patten Jennifer, Halligan Patrick, Bashiri Ghazal, Kegel Michael, Bonadio Jacob D, Wang Karin
Department of Bioengineering, Temple University, Pennsylvania.
bioRxiv. 2024 Sep 25:2024.09.23.614581. doi: 10.1101/2024.09.23.614581.
Fibronectin (Fn) is an extracellular matrix glycoprotein with mechanosensitive structure-function. EDA Fn, a Fn isoform, is not present in adult tissue but is required for tissue repair. Curiously, EDA Fn is linked to both regenerative and fibrotic tissue repair. Given that Fn mechanoregulates cell behavior, Fn EDA organization during wound closure might play a role in mediating these differing responses. One mechanism by which cells sense and respond to their microenvironment is by activating a transcriptional co-activator, Yes-associated protein (YAP). Interestingly, YAP activity is not only required for wound closure, but similarly linked to both regenerative and fibrotic repair. Therefore, this study aims to evaluate how, during normal and fibrotic wound closure, EDA Fn organization might modulate YAP translocation by culturing human dermal fibroblasts on polydimethylsiloxane (PDMS) substrates mimicking normal (soft: 18 kPa) and fibrotic (stiff: 146 kPa) wounded skin. On stiffer substrates mimicking fibrotic wounds, fibroblasts assembled an aligned EDA Fn matrix comprising thinner fibers, suggesting increased microenvironmental tension. To evaluate if cell binding to the EDA domain of Fn was essential to overall matrix organization, fibroblasts were treated with Irigenin, which inhibits binding to the EDA domain within Fn. Blocking adhesion to EDA led to randomly organized EDA Fn matrices with thicker fibers, suggesting reduced microenvironmental tension even during fibrotic wound closure. To evaluate if YAP signaling plays a role in EDA Fn organization, fibroblasts were treated with CA3, which suppresses YAP activity in a dose-dependent manner. Treatment with CA3 also led to randomly organized EDA Fn matrices with thicker fibers, suggesting a potential connected mechanism of reducing tension during fibrotic wound closure. Next, YAP activity was assessed to evaluate the impact of EDA Fn organization. Interestingly, fibroblasts migrating on softer substrates mimicking normal wounds increased YAP activity but on stiffer substrates, decreased YAP activity. When fibroblasts on stiffer substrates were treated with Irigenin or CA3, fibroblasts increased YAP activity. These results suggest there may be disrupted signaling between EDA Fn organization and YAP translocation during fibrotic wound closure that could be restored when reestablishing normal EDA Fn matrix organization to instead drive regenerative wound repair.
纤连蛋白（Fn）是一种具有机械敏感结构功能的细胞外基质糖蛋白。EDA Fn是Fn的一种同工型，在成年组织中不存在，但在组织修复中是必需的。奇怪的是，EDA Fn与再生性和纤维化组织修复都有关联。鉴于Fn对细胞行为具有机械调节作用，伤口闭合过程中Fn EDA的组织形式可能在介导这些不同反应中发挥作用。细胞感知并响应其微环境的一种机制是通过激活一种转录共激活因子，即Yes相关蛋白（YAP）。有趣的是，YAP活性不仅是伤口闭合所必需的，而且同样与再生性和纤维化修复相关。因此，本研究旨在通过在模仿正常（柔软：18千帕）和纤维化（坚硬：146千帕）伤口皮肤的聚二甲基硅氧烷（PDMS）底物上培养人真皮成纤维细胞，来评估在正常和纤维化伤口闭合过程中，EDA Fn的组织形式如何调节YAP易位。在模仿纤维化伤口的较硬底物上，成纤维细胞组装了一个由较细纤维组成的排列整齐的EDA Fn基质，这表明微环境张力增加。为了评估细胞与Fn的EDA结构域结合对整体基质组织是否至关重要，用艾瑞宁处理成纤维细胞，艾瑞宁可抑制与Fn内EDA结构域的结合。阻断与EDA的黏附导致形成随机组织的、纤维更粗的EDA Fn基质，这表明即使在纤维化伤口闭合过程中微环境张力也降低了。为了评估YAP信号传导在EDA Fn组织形成中是否起作用，用CA3处理成纤维细胞，CA3以剂量依赖的方式抑制YAP活性。用CA3处理也导致形成随机组织的、纤维更粗的EDA Fn基质，这表明在纤维化伤口闭合过程中存在一种潜在的降低张力的关联机制。接下来，评估YAP活性以评估EDA Fn组织形成的影响。有趣的是，在模仿正常伤口的较软底物上迁移的成纤维细胞YAP活性增加，但在较硬底物上YAP活性降低。当用艾瑞宁或CA3处理在较硬底物上的成纤维细胞时，成纤维细胞YAP活性增加。这些结果表明，在纤维化伤口闭合过程中，EDA Fn组织形成与YAP易位之间的信号传导可能被破坏，而当重新建立正常的EDA Fn基质组织以驱动再生性伤口修复时，这种信号传导可能会恢复。
