ECM prestrain generated by myofibroblast contraction affects TGF-β1 activation. Fibroblasts secrete ECM that is rich in FN (green) and the large latent complex of TGF-β1 (LTBP-1, LAP, and TGF-β1). Fibroblast-to-myofibroblast differentiation occurs during physiological (normal wound healing) and pathological (fibrosis) tissue remodeling. Myofibroblasts are characterized by α-SMA–positive stress fibers (red) enabling these cells to exert high contractile activity and forces transmitted to the ECM at sites of integrins. The gradual straitening and straining of ECM fibrils, containing FN and LTBP-1, prime the latent TGF-β1 complex for subsequent activation. At sufficient prestrain, minimal additional length changes in the ECM (i.e., small contractions) will be sufficient to release active TGF-β1 by inducing a conformational change in LAP. Hence, the mechanical preloading of the ECM determines the trigger point for TGF-β1 activation driving the vicious loop of myofibroblast self-activation.