Boris Hinz

The conjunctive presence of mechanical stress and active transforming growth factor beta1 (TGF-beta1) is essential to convert fibroblasts into contractile myofibroblasts, which cause tissue contractures in fibrotic diseases. Using cultured myofibroblasts and conditions that permit tension modulation on the extracellular matrix (ECM), we establish that myofibroblast contraction functions as a mechanism to directly activate TGF-beta1 from self-generated stores in the ECM. Contraction of myofibroblasts and myofibroblast cytoskeletons prepared with Triton X-100 releases active TGF-beta1 from the ECM. This process is inhibited either by antagonizing integrins or reducing ECM compliance and is independent from protease activity. Stretching myofibroblast-derived ECM in the presence of mechanically apposing stress fibers immediately activates latent TGF-beta1. In myofibroblast-populated wounds, activation of the downstream targets of TGF-beta1 signaling Smad2/3 is higher in stressed compared to relaxed tissues despite similar levels of total TGF-beta1 and its receptor. We propose activation of TGF-beta1 via integrin-mediated myofibroblast contraction as a potential checkpoint in the progression of fibrosis, restricting autocrine generation of myofibroblasts to a stiffened ECM.

To evaluate whether alpha-smooth muscle actin (alpha-SMA) plays a role in fibroblast contractility, we first compared the contractile activity of rat subcutaneous fibroblasts (SCFs), expressing low levels of alpha-SMA, with that of lung fibroblasts (LFs), expressing high levels of alpha-SMA, with the use of silicone substrates of different stiffness degrees. On medium stiffness substrates the percentage of cells producing wrinkles was similar to that of alpha-SMA-positive cells in each fibroblast population. On high stiffness substrates, wrinkle production was limited to a subpopulation of LFs very positive for alpha-SMA. In a second approach, we measured the isotonic contraction of SCF- and LF-populated attached collagen lattices. SCFs exhibited 41% diameter reduction compared with 63% by LFs. TGFbeta1 increased alpha-SMA expression and lattice contraction by SCFs to the levels of LFs; TGFbeta-antagonizing agents reduced alpha-SMA expression and lattice contraction by LFs to the level of SCFs. Finally, 3T3 fibroblasts transiently or permanently transfected with alpha-SMA cDNA exhibited a significantly higher lattice contraction compared with wild-type 3T3 fibroblasts or to fibroblasts transfected with alpha-cardiac and beta- or gamma-cytoplasmic actin. This took place in the absence of any change in smooth muscle or nonmuscle myosin heavy-chain expression. Our results indicate that an increased alpha-SMA expression is sufficient to enhance fibroblast contractile activity.