Fetal and postnatal sera differentially modulate human dermal fibroblast phenotypic and functional features in vitro

Abstract

Fetal wounds heal without scar formation, fibrosis, or contracture. Compared with adult wounds, they are characterized by major differences in the extracellular matrix and the absence of myofibroblastic cells. The reasons for these differences are not well known and determination of factors affecting the absence of scarring in the fetus may lead to strategies for controlling adult pathological scarring. In the present study, we have assessed the effects of serum on the behavior of normal human dermal fibroblasts. Using an in vitro approach, we investigated the effects of fetal and adult serum on cell properties such as growth rate, collagen synthesis, gelatinase activities, and differentiation to myofibroblasts using biochemical, morphological, and ultrastructural parameters. We studied the induction of alpha-smooth muscle (alpha-SM) actin in fibroblasts, and its correlation with increased collagen gel contraction by the cells. Our results showed that, compared with FBS (fetal bovine serum), postnatal calf serum (PCS) decreased mitogenic activity and collagenase synthesis but not collagen synthesis. Furthermore, cells cultured with PCS differentiated to myofibroblasts with an increase in cell diameter, number of stress fibers, alpha-SM actin expression, and collagen gel contraction. To characterize the molecules involved in this differentiation process, the amount of transforming growth factor beta (TGFbeta) in FBS and PCS was determined and the effect of neutralizing anti-TGFbeta antibody was evaluated. It was determined that FBS contained more TGFbeta than PCS, but that essentially all the TGFbeta was latent in both sera. However, results obtained with anti-TGFbeta antibody show that active TGFbeta is present when human dermal fibroblasts are cultured with medium containing PCS. These results suggest that, in the presence of PCS but not FBS, the cells either produce active TGFbeta or an enzyme that is able to activate latent serum TGFbeta. Alternatively, sera may contain two different forms of latent TGFbeta, the PCS form being activated by the dermal fibroblast cells. A similar mechanism may be involved, at least in part, in skin wound healing and may underlie the appearance of myofibroblasts in postnatal wounds.

Increased collagen production in fibroblasts cultured from irradiated skin and effect of TGF β1– clinical study

“…It was observed consistently in cell lines from all 5 patients studied, though the magnitude of the difference was variable. Because of the standardization of experimental conditions in this study, the observed elevation of collagen synthesis rates in irradiated cells when compared to controls cannot be explained on the basis of differences in variables known potentially to affect the functional characteristics of fibroblasts in culture, such as passage number (E1 Nabout et al, 1989) or local cytokine environment (Moulin et al, 1997). Our demonstration of increased collagen production rates in cells cultured from areas irradiated in vivo is consistent with the finding of Kolb et al (1998) of a radiation dose-dependent increase in collagen production (as measured by an ELISA estimation of the concentration of collagen I propeptide) by human lung fibroblasts irradiated in vitro.…”

Rapid and dynamic regulation of TGF-β receptors on blood vessels and fibroblasts during ischemia-reperfusion injury

“…In comparison studies, this antibody displayed the same specificity as the antibodies obtained from Dr. A. Moustakas (Whitehead Institute for Medical Research, Cambridge, MA). The procedure for the preparation of anti-TGF-␤1 antibody involved coupling of TGF-␤1 to keyhole limpet hemocyanin and injection of rabbits and was described previously (25). Normal rabbit IgG used as a negative control was obtained from Lipshaw Immunon (Pittsburgh, PA).…”

Expression of Heat Shock Proteins in Mouse Skin During Wound Healing

“…Indirect immunofluorescence labeling of frozen sections (fixed for 10 min in Ϫ20C methanol) was realized using polyclonal anti-keratin 6 (1:500), Hsp60 (1:100), Hsp90 (1:100), Hsp27 (1:100), and Hsp70 (1:100) antibodies and rhodamine-conjugated secondary antibody (1:100) as described previously (Moulin et al 1997). In controls, normal rabbit serum was used at the same dilution as the primary antibody.…”