Jeffrey C. Geesin

Retinoids affect collagen synthesis through inhibition of ascorbate-induced lipid peroxidation in cultured human dermal fibroblasts☆

Ascorbate has been shown to stimulate collagen synthesis in cultured human dermal fibroblasts by increasing transcription of the collagen genes. In the present studies, ascorbate stimulates lipid peroxidation at concentrations similar to those necessary to affect collagen synthesis. Molecules which inhibit lipid peroxidation, such as propyl gallate, cobalt chloride, and alpha-naphthol, also inhibit collagen synthesis, suggesting a correlation between the two phenomena. Retinoic acid and some synthetic retinoids have previously been shown to inhibit collagen synthesis in cultured human dermal fibroblasts. In our studies two different retinoids, at similar concentrations, inhibit both ascorbate-stimulated lipid peroxidation and collagen synthesis. Since high concentrations of retinoids were required, the ability of retinoids to inhibit the oxidant effect of ascorbate, and not their receptor-mediated activity, may be responsible for their effect on collagen synthesis.

Development of a Three-Dimensional Transmigration Assay for Testing Cell–Polymer Interactions for Tissue Engineering Applications

The ability of synthetic or natural scaffolds to support invasion of cells from surrounding tissue is a key parameter for tissue engineering (TE). In this study, the migration of fibroblasts, chondrocytes, and osteoblasts into biodegradable polymer scaffolds was evaluated using a novel, three-dimensional (3-D) transmigration assay. This assay is based on a cell-populated contracted collagen lattice with a biodegradable polymer scaffold implanted at the center of the collagen gel. Cell migration into the scaffolds was assessed both quantitatively and qualitatively following various time lengths in culture using image analysis. Chondrocytes, incorporated within the collagen lattice, migrated into polymer scaffolds, when cultured both statically or in a rotating bioreactor. However, the bioreactor cultures resulted in a significantly greater cell invasion as compared to static cultures. There was a cell density-dependent osteoblast migration from collagen lattice into polymer scaffold, when tested in the transmigration assay. In addition, polymer scaffolds, treated with or without recombinant human platelet-derived growth factor (rh-PDGF-BB) were evaluated for fibroblast migration. The presence of rh-PDGF-BB resulted in significantly greater fibroblast invasion as compared to untreated scaffolds. Our studies suggest that the transmigration model provides a rapid system for testing cell invasion of potential scaffolds for tissue engineering applications.

Regulation of collagen synthesis by ascorbic acid: Characterization of the role of ascorbate-stimulated lipid peroxidation

Recently, we have described the ability of traditional lipid peroxidation inhibitors to inhibit ascorbate-stimulated collagen synthesis. In order to characterize further this effect, we have tested the ability of known and potential inhibitors of lipid peroxidation for their effects on ascorbate-stimulated collagen synthesis and lipid peroxidation. In our experiments, mannitol, a water soluble antioxidant, had no effect on ascorbate-induced collagen synthesis nor on lipid peroxidation. However, alpha-tocopherol, which is a lipophilic antioxidant, inhibited both effects of ascorbate. Superoxide dismutase, catalase, and their polyethylene glycol conjugate forms did not inhibit the ascorbate-stimulated collagen synthesis or lipid peroxidation. In addition, no effect was seen with the oxygen radical scavengers isopropanol, ethanol, or dimethyl sulfoxide. Two iron chelators, o-phenanthroline and alpha,alpha-dipyridyl, both inhibited ascorbate-induced lipid peroxidation and collagen synthesis, consistent with the previously described iron-dependence of lipid peroxidation by ascorbate. These results support a correlation between collagen synthesis and lipid peroxidation and provide a theory for the mechanism of ascorbic acid regulation of collagen synthesis.

Differential expression and regulation of extracellular matrix-associated genes in fetal and neonatal fibroblasts

Adults and neonates heal wounds by a repair process associated with scarring in contrast to scar‐free wound healing in the fetus. In the present study, human dermal fetal fibroblasts, representing the scarless phenotype, and neonatal human dermal fibroblasts, representing scar‐forming phenotype, were examined for potential differences that might influence the wound healing process. Fetal fibroblasts secreted four‐ to tenfold more latent transforming growth factor‐β1 depending on the cell strains compared. Fetal fibroblasts also produced higher levels of collagen protein and mRNA for most types of collagen (particularly type III) as compared to neonatal cells. Interestingly, mRNA for type V collagen was significantly reduced in fetal cells. Neonatal fibroblasts expressed significantly higher levels of latent transforming growth factor‐β1 binding protein mRNA, in contrast to almost undetectable levels in fetal fibroblasts. By ligand blot analysis, the levels of insulin‐like growth factor binding protein‐3, a reported mediator of transforming growth factor‐β1 activity, was eightfold higher in neonatal versus fetal fibroblasts. Approximately 20 other mRNAs for various cytokines, matrix molecules and receptors were examined and found to be similar between the two cell types. The phenotypic differences described in this article may represent potentially important mechanisms to explain the differences in the quality of wound repair observed in fetal versus adult/neonatal tissues.

Modulation of collagen synthesis by growth factors : the role of ascorbate-stimulated lipid peroxidation

Ascorbic acid has been shown to stimulate collagen synthesis through induction of lipid peroxidation leading to increased transcription of the collagen genes. The mechanism by which lipid peroxidation stimulates collagen transcription is unknown; however, an alteration of cell membranes may affect the activity of serum growth factors leading to a change in gene expression. To test this hypothesis, we treated dermal fibroblasts with transforming growth factor-beta (TGF-beta), epidermal growth factor (EGF), interleukin-1 (IL-1), platelet-derived growth factor (PDGF), or fibroblast growth factor (FGF) in the presence of lipid peroxidation stimulating (200 microM) and nonstimulating (1 microM) concentrations of ascorbic acid. EGF and IL-1 had no effect on collagen synthesis at either concentration of ascorbic acid. FGF affected collagen synthesis only in the presence of 200 microM ascorbic acid, producing both a stimulation (0.4-2 ng/ml) and an inhibition (greater than 50 ng/ml). PDGF and TGF-beta stimulated collagen synthesis in the presence of both concentrations of ascorbic acid, with TGF-beta producing an 11-fold increase in collagen synthesis in the presence of ascorbate. This synergism produced by the combination of ascorbic acid and TGF-beta was inhibitable by the lipid peroxidation inhibitor, propyl gallate. These results indicate that regulation of collagen synthesis by ascorbic acid does not occur through altering the response to EGF or Il-1. Ascorbate has no effect on PDGF but the effects of TGF-beta and FGF on collagen synthesis appear to be sensitive to lipid peroxidation.

The differential regulation and secretion of proteinases from fetal and neonatal fibroblasts by growth factors

One of the major differences between fetal and adult wound repair is the unique ability of fetal wounds to heal without scarring. Since scar formation is a function of extracellular matrix deposition, the regulation of this component is fundamental in tissue remodeling. In this study, we have characterized the differences in the secretion of matrix-degrading proteases, namely urokinase plasminogen activator and gelatinase A and B, from fetal and neonatal fibroblasts. In addition, we examined the modulation of these protease levels by growth factors known to be important in wound repair. The results indicate that the secretion of these proteases differ significantly between the two cell types. The levels of urokinase plasminogen activator and its inhibitor were notably higher in media conditioned by neonatal fibroblasts in comparison to fetal samples. In contrast, the basal level of gelatinase A was comparable in both cell types, whilst the level of gelatinase B was elevated in the fetal fibroblasts. Transforming growth factor-beta 1 reduced the level of urokinase plasminogen activator and stimulated the secretion of plasminogen activator inhibitor-1 and progelatinase B in both neonatal and fetal fibroblasts. However, only progelatinase A and an activated form of gelatinase B were significantly elevated in fetal fibroblasts. In contrast, platelet-derived growth factor stimulated urokinase plasminogen activator, its inhibitor and both gelatinase A and B, an effect which was more apparent in fetal fibroblasts. This difference in protease regulation may be reflected in the differing rate and quality of tissue remodeling observed during adult vs fetal wound repair.

Development of a skin model based on insoluble fibrillar collagen

A biocompatible, 3-dimensional, noncontracting, crosslinked collagen matrix was adapted to promote differentiation of epidermal keratinocytes. To produce the matrix, a 3% wt/wt dispersion of insoluble bovine collagen containing 5 mg polylysine/g collagen in 0.001 N HCl was blended, lyophilized, and crosslinked using a dehydrothermal technique. Matrices 4 cm2 and 3 mm thick were seeded with human dermal fibroblasts (1 x 10(5)/cm2). After 5 days in culture, the matrices were seeded with human epidermal keratinocytes (1 x 10(5)/cm2). The cultures were grown submerged for 1 week and raised to the liquid/air interface for 3 weeks to promote epidermal differentiation. Based on morphology and immunological staining with antibodies for human involucrin, keratin 1 (k1), filaggrin, and loricrin, the state of differentiation of the epidermal layer was nearly equivalent to that seen with cultures grown on contracted collagen lattices produced according to the methodology described in the literature and similar to the pattern produced in normal neonatal foreskin. These results demonstrate the usefulness of an in vitro skin model employing a crosslinked collagen matrix that permits the incorporation of additional covalently linked bioactive molecules during matrix formation.

Characterization of a macrophage-based system for studying the activiation of latent TGF-β

TGF-β has been implicated in scarring and tissue fibrosis. Most cells secrete TGF-β 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- β1 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-β. The activation mechanism in our system involves mannose 6-phosphate/insulin-like growth factor-II receptor andtransglutaminase. The activation of latent TGF-β 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-β. Anti-inflammatory molecules, such as hydrocortisone, when added to LPS-treated macrophages, inhibited TGF-β 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-β activation, thus providing a screening method for potential anti-scarring molecules.