Myofibroblast contraction activates latent TGF-β1 from the extracellular matrix. Myofibroblasts, which are contractile cells, generate tension in the extracellular matrix (ECM) and are essential for tissue repair. However, excessive myofibroblast activity contributes to fibrosis in various organs. This study shows that myofibroblast contraction directly activates latent TGF-β1 stored in the ECM. Contraction of myofibroblasts and their cytoskeletons releases active TGF-β1 from the ECM, and this process is inhibited by antagonizing integrins. The study also demonstrates that mechanical stress and TGF-β1 signaling are crucial for myofibroblast differentiation. TGF-β1 is secreted as part of a large latent complex (LLC), which includes latent TGF-β1, LAP, and LTBP-1. The LLC binds to other ECM components, such as fibrillin-1 and fibronectin. Activation of latent TGF-β1 can occur through proteolytic cleavage by proteases or through integrin-mediated traction. The study shows that myofibroblast contraction activates latent TGF-β1 by transmitting mechanical stress to the LLC, which leads to conformational changes in LAP and the release of active TGF-β1. This process is dependent on α-SMA-positive stress fibers and integrin binding to LAP in the LLC. The study also shows that ECM stiffness modulates the level of TGF-β1 release by contraction, restricting autocrine maintenance of the myofibroblast phenotype to the appropriate mechanical microenvironment. The findings suggest that mechanical tension and TGF-β1 signaling are essential for myofibroblast differentiation and that myofibroblast contraction activates latent TGF-β1 from the ECM. The study also shows that the efficiency of latent TGF-β1 activation by myofibroblast contraction increases with increasing ECM stiffness. The results indicate that the transmission of myofibroblast force to the LLC induces a conformational change in LAP, leading to the release of active TGF-β1. The study also shows that the activation of latent TGF-β1 by myofibroblast contraction is dependent on integrin binding to LAP in the LLC. The findings suggest that the mechanical properties of the ECM are crucial for the activation of latent TGF-β1 by myofibroblast contraction. The study also shows that the activation of latent TGF-β1 by myofibroblast contraction is dependent on the presence of a mechanoresistant ECM. The results indicate that the mechanical tension generated by myofibroblast contraction is essential for the activation of latent TGF-β1 from the ECM. The study also shows that the activation of latent TGF-β1 by myofibroblast contraction is dependent onMyofibroblast contraction activates latent TGF-β1 from the extracellular matrix. Myofibroblasts, which are contractile cells, generate tension in the extracellular matrix (ECM) and are essential for tissue repair. However, excessive myofibroblast activity contributes to fibrosis in various organs. This study shows that myofibroblast contraction directly activates latent TGF-β1 stored in the ECM. Contraction of myofibroblasts and their cytoskeletons releases active TGF-β1 from the ECM, and this process is inhibited by antagonizing integrins. The study also demonstrates that mechanical stress and TGF-β1 signaling are crucial for myofibroblast differentiation. TGF-β1 is secreted as part of a large latent complex (LLC), which includes latent TGF-β1, LAP, and LTBP-1. The LLC binds to other ECM components, such as fibrillin-1 and fibronectin. Activation of latent TGF-β1 can occur through proteolytic cleavage by proteases or through integrin-mediated traction. The study shows that myofibroblast contraction activates latent TGF-β1 by transmitting mechanical stress to the LLC, which leads to conformational changes in LAP and the release of active TGF-β1. This process is dependent on α-SMA-positive stress fibers and integrin binding to LAP in the LLC. The study also shows that ECM stiffness modulates the level of TGF-β1 release by contraction, restricting autocrine maintenance of the myofibroblast phenotype to the appropriate mechanical microenvironment. The findings suggest that mechanical tension and TGF-β1 signaling are essential for myofibroblast differentiation and that myofibroblast contraction activates latent TGF-β1 from the ECM. The study also shows that the efficiency of latent TGF-β1 activation by myofibroblast contraction increases with increasing ECM stiffness. The results indicate that the transmission of myofibroblast force to the LLC induces a conformational change in LAP, leading to the release of active TGF-β1. The study also shows that the activation of latent TGF-β1 by myofibroblast contraction is dependent on integrin binding to LAP in the LLC. The findings suggest that the mechanical properties of the ECM are crucial for the activation of latent TGF-β1 by myofibroblast contraction. The study also shows that the activation of latent TGF-β1 by myofibroblast contraction is dependent on the presence of a mechanoresistant ECM. The results indicate that the mechanical tension generated by myofibroblast contraction is essential for the activation of latent TGF-β1 from the ECM. The study also shows that the activation of latent TGF-β1 by myofibroblast contraction is dependent on