Myofibroblast TGF-ß Activation Measurement In Vitro.

ABSTRACT

Myofibroblasts are critical to processes involved in normal wound healing and during pathological fibrosis. They transdifferentiate from fibroblasts, and in doing so become contractile and capable of secreting large amounts of extracellular matrix proteins. Transforming growth factor-beta (TGFß) is a key cytokine involved in wound healing and fibrogenesis. TGFß signaling has long been the subject of experimental therapeutic approaches to inhibit fibrosis in a variety of organ systems. Inhibition of TGFß can reduce myofibroblast transdifferentiation, contractility, and matrix production. Importantly, TGFß is released from cells and sequestered in the extracellular matrix in a latent form that requires activation for biological function. There have been multiple mechanisms of TGFß activation described in a variety of cell types and in cell free systems; however, myofibroblasts have previously been shown to activate TGFß via cell surface integrins, particularly αvß5 integrins. This chapter will provide detailed protocols for accurately measuring activation of TGFß by myofibroblasts in vitro. Levels of active TGFß usually represent a small proportion of the total amount of latent TGFß present in the matrix. Methods to measure active TGFß therefore need to be sensitive and specific to detect the active cytokine only.