Characterization of a macrophage-based system for studying the activation of latent TGF-beta.

TGF-beta has been implicated in scarring and tissue fibrosis. Most cells secrete TGF-beta as a high molecular weight, latent complex that must be processed to a lower molecular weight, biologically active form. A number of molecules are involved in this activation step including the mannose 6-phosphate/insulin-like growth factor-II receptor, tissue transglutaminase, thrombospondin, plasmin, and others. Here we describe a rapid macrophage-based system for TGF-beta1 activation, which could be used for screening potential anti-fibrotic agents. The system employs transformed mouse peritoneal macrophages treated with lipopolysaccharide as a cell line capable of activating latent TGF-beta. The activation mechanism in our system involves mannose 6-phosphate/insulin-like growth factor-II receptor and transglutaminase. The activation of latent TGF-beta in this system can be prevented by the addition of mannose-6-phosphate but not mannose-1-phosphate. In addition, transglutaminase inhibitors, antibodies to thrombospondin, insulin-like growth factor-II in the presence of its binding protein IGFBP-2, but not IGFBP-1, suppressed the activation of TGF-beta. Anti-inflammatory molecules, such as hydrocortisone, when added to LPS-treated macrophages, inhibited TGF-beta activation by a mechanism, that may involve downregulation of transglutaminase expression. In summary, this new, rapid and reproducible system allows testing molecules for their ability to inhibit TGF-beta activation, thus providing a screening method for potential anti-scarring molecules.

Regulation of collagen synthesis in human dermal fibroblasts by the sodium and magnesium salts of ascorbyl-2-phosphate.

Ascorbic acid has been shown to stimulate collagen synthesis in dermal fibroblasts by increasing the rate of transcription of collagen genes. Experiments involving the use of ascorbic acid require daily supplementation due to the instability of the molecule in aqueous solutions. In order to provide a more stable alternative to ascorbic acid, two salts of ascorbyl-2-phosphate, having a greater chemical stability than ascorbic acid, were tested for their ability to stimulate collagen synthesis in monolayer fibroblast cultures. The concentration and time dependence of their activities were compared with ascorbic acid. The magnesium salt of ascorbyl-2-phosphate was found to be equivalent to ascorbic acid in stimulating collagen synthesis in these assays, while the sodium salt required at least a tenfold greater concentration to produce the same effect as ascorbic acid. Solutions of either ascorbic acid or the ascorbyl-2-phosphate analogs (at 10 mM) in phosphate-buffered saline (PBS) were relatively stable as shown by their decay rates and their ability to stimulate collagen synthesis even after nine days in solution prior to testing their effects on cultured cells. Ascorbic acid was unstable at neutral pH compared to solutions of either sodium or magnesium ascorbyl-2-phosphate. These data support the use of magnesium ascorbyl-2-phosphate in experiments where stability of ascorbic acid is a concern, e.g. in long-term cultures or in in vivo studies.

Ascorbic acid stimulates collagen production without altering intracellular degradation in cultured human skin fibroblasts.

The influence of ascorbic acid on intracellular degradation of collagen synthesized by cultured human-skin fibroblasts was examined. In confluent cells maintained in 0.5% serum-supplemented medium, ascorbic acid had no significant effect on collagen degradation measured with hydroxyproline as the marker. Similar results were obtained when collagen degradation was measured with the marker hydroxylysine, the cellular synthesis of which is independent of ascorbic acid. The stimulatory effects of ascorbic acid on collagen production therefore cannot be explained by a change in the rate of degradation. Ascorbic acid acts at some as yet undetermined level to increase the rate of collagen synthesis.