Epidermal growth factor (EGF) is a potent mitogen in vitro, but its biological role is less clear. The vulnerary effects of EGF were evaluated in a model of wound repair, the polyvinyl alcohol sponge implanted subcutaneously in rats. EGF was purified to homogeneity by reverse-phase HPLC and quantified by receptor binding assay and amino acid analysis. Preliminary data showed moderate promotion of granulation tissue formation by daily injections of 10 /Ag of EGF. To test the hypothesis that long-term exposure to EGF is required for complete cellular response, the factor was incorporated into pellets releasing 10 or 20 jig of biologically active EGF per day, and the pellets were embedded within the sponges. Slow release of EGF caused a dramatic increase in the extent and organization of the granulation tissue at day 7, a doubling in the DNA content, and 33% increases in protein content and wet weight, as compared with placebo controls. Although collagen content was also increased by almost 50%, the relative rate of collagen synthesis remained the same, suggesting that the morphological and biochemical increase in coflagen resulted from increased numbers of fibroblasts rather than a specific stimulation of collagen synthesis. These results indicate that the local sustained presence ofEGIF accelerates the process of wound repair, specifically neovascularization, organization by fibroblasts, and accumulation of collagen.Epidermal growth factor (EGF) stimulates a variety of biological phenomena, including proliferation of skin and corneal epithelia in organ culture, proliferation and differentiation of epidermis and corneal epithelial cells in vivo (1), neoangiogenesis in the rabbit cornea (2), and the synthesis of DNA, RNA, protein, and hyaluronic acid in various cell lines in culture (1). Many cell types, including dermal fibroblasts, possess EGF surface receptors and will proliferate in response to EGF in cell culture (3). By contrast to the many observations in vitro, the function of growth factors during wound repair is not clear. This is in part the result of the difficulty of delivering growth factors and quantifying the responses to them in vivo.The process of healing surface wounds includes reepithelization, neovascularization, granulation tissue development, collagen elaboration, maturation and remodeling of the scar, and contraction (4). During the early phases of repair, the local accumulation of collagen strongly correlates with the accretion of tensile strength (5); hence, measuring the content and concentration of collagen at a repair site permits an estimate of the rate of healing.
Cartilage-derived growth factor (CDGF), a cationic polypeptide of ~18,000 mol wt, was prepared from bovine articular cartilage; other sources were bovine and human scapular and costal cartilage. Previous studies have shown that CDGF stimulates the proliferation of cultured mouse fibroblasts as well as chondrocytes and endothelial cells from various sources. In this study, CDGF was shown to stimulate dose-dependently the accumulation of DNA and collagen by rat embryo fibroblasts and a population of fibroblasts derived from granulation tissue. CDGF also stimulated the proliferation of cultured bovine capillary endothelial cells dose-dependently. To evaluate the effects of CDGF in vivo, we implanted polyvinyl alcohol sponges subcutaneously in rats. 6 d postimplantation, sponges were injected with 300 pg of partially purified CDGF, a dose which takes into account the cell numbers in the sponges as compared with cell cultures. CDGF rapidly disappeared from the sponges and only ~10% of the initial dose was present at 4 h. Despite its transient presence, CDGF caused a relative increase in sponge DNA content of 2.6-fold at 48 h and 2.4-fold at 72 h. We repeated the sponge experiment by using 500-ng injections of CDGF purified to near homogeneity by heparin-Sepharose chromatography. Purified CDGF caused significant increases in sponge collagen, protein, and DNA content at 48 and 72 h after a single injection. The effects of CDGF were abolished by heat and unaffected by reduction of disulfide linkages. Morphologically, CDGF did not evoke an inflammatory response, and its effect on proliferating endothelial cells and fibroblasts was, therefore, probably direct. However, increases in DNA content of sponges could not be fully accounted for by increased DNA synthesis, which suggests that recruitment may be an important component of the in vivo response. Taken together, the effects of CDGF on cultured cells and granulation tissue suggest that the sustained presence of CDGF in vivo may greatly enhance its effects upon wound repair.A number of polypeptide growth factors that stimulate the proliferation of cells in culture have been described in the last decade. These include epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor (5,6,17). Many of the growth factors are large polypeptides that have specific receptor-mediated interactions with their target cells, and such interactions are associated with cell proliferation in vitro. By contrast, the efficacy of growth factors in vivo is not nearly so clearly established. This is in part the result of the difficulty of administering growth factors and measuring their cellular response in vivo.The cartilage-derived growth factor (CDGF)' is a cationic polypeptide with a molecular weight of ~ 18,000 which can 1 Abbreviations used in this paper. BCE, bovine capillary endothelial; CDGF, cartilage-derived growth factor; DME, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; TCA, trichloroacetic acid.
Keratinocytes produce a molecule, epidermal-derived thymocyte activating factor (ETAF), which is biologically and physiochemically similar to the polypeptide hormone interleukin 1 (IL-1). Because the stratum corneum (SC) is composed of terminally differentiated keratinocytes, we questioned whether ETAF/IL-1 could be isolated from this tissue. The extraction of normal human SC with a physiologic saline solution yielded a large amount of ETAF/IL-1 activity, as measured by the in vitro thymocyte co-stimulator assay. SC-derived ETAF/IL-1 (scETAF/IL-1) eluted from a sizing column with an approximate molecular weight of 15,000, and demonstrated three isoelectric point forms after separation on a chromatofocusing column. By these physiochemical characteristics, scETAF/IL-1 was found to be similar, if not identical to human keratinocyte-and macrophage-derived ETAF/IL-1. Further, a number of biologic effects known to occur in vivo after the administration of ETAF/ IL-1, such as fever, neutrophilia, and an increase in plasma levels of acute-phase proteins, were all induced by the injection of scETAF/IL-1 into endotoxin-nonresponsive mice. scETAF/IL-1 was also found to stimulate collagenase production by human fibroblasts in vitro. In summary, our studies have established that normal human SC contains a large quantity of scETAF/IL-1. Whether scETAF/IL-1 integrates into the earliest afferents phases of local inflammatory responses, or merely represents a means of disposal of excessively produced hormone is currently unresolved.
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