THE MYOFIBROBLAST MARKERS ALPHA-SM ACTIN AND BETA-ACTIN ARE DIFFERENTIALLY EXPRESSED IN 2-D AND 3-D CULTURE MODELS OF FIBROTIC AND NORMAL SKIN

To characterize the differences between fibrotic myofibroblasts and no rmal fibroblasts, we studied two differentiation markers: alpha-smooth muscle (SM) actin, a specific marker of myofibroblast differentiation , and beta-actin, which is overexpressed in the fibrotic tissue. Exper iments were performed on fibroblasts isolated from normal pig skin and on subcutaneous myofibroblasts isolated from pig radiation-induced fi brosis. Three culture models were used: cells in monolayers, equivalen t dermis, consisting of fibroblasts embedded into a matrix composed of type I collagen, and in vitro reconstituted skin, in which the matrix and containing life fibroblasts were overlaid with keratinocytes. Sam ples were studied using immunofluorescence and western-blotting. In mo nolayers cultures, both fibrosis and normal cells expressed alpha-SM a ctin. Furthermore, similar amounts of beta-actin protein were found. I n these conditions, the resulting alterations in the phenotypes of cel ls made comparison of cultured fibrotic and normal cells irrelevant. U nder the two 3-D culture models, normal fibroblasts no longer expresse d alpha-SM actin. They expressed beta-actin at the basal level. Moreov er, the fibrotic myofibroblasts in both 3-D models retained their diff erentiation features, expressing alpha-SM actin and overexpressing bet a-actin. We found that this normalization was mainly related to the ge nomic programmation acquired by the cells in the tissue. Cellular moti lity and microenvironment were also involved, whereas cellular prolife ration was not a major factor. Consequently, both three-dimensional mo dels allowed the study of radiation-induced fibrosis in vitro, provide d good extrapolations to in vivo conditions and avoided certain of cul ture artefacts.