David A. Belford

Milk-derived growth factors as serum supplements for the growth of fibroblast and epithelial cells

SummaryWe have investigated the response of several epithelial and fibroblastic cells to a mitogenic extract of bovine milk. Cation exchange chromatography was used to produce a mitogen-rich fraction from an industrial whey source that, although comprising only 0.5% of total whey protein, contained the bulk of the growth factor activity. This fraction was a source of potent growth promoting activity for all mesodermal-derived cells tested, including human skin and embryonic lung fibroblasts, Balb/c 3T3 fibroblasts, and rat L6 myoblasts. Maximal growth of all these cell types exceeded that observed in 10% fetal bovine serum. Feline kidney and baby hamster fibroblasts and Chinese hamster ovary cells were less responsive, achieving a maximal growth response of 50–75% that observed in 10% fetal bovine serum. Maximal growth achieved in whey-extract-supplemented cultures of Balb/c 3T3 and human skin fibroblasts, and L6 myoblast cultures exceeded that seen in response to recombinant acidic or basic fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, or epidermal growth factor. Importantly, addition of low concentrations of fetal bovine serum to the whey-derived mitogenic fraction produced an additive response. However, concentrated milk-derived factors were found to be inhibitory to the growth of all epithelial lines tested, including rat intestinal epithelial cells, canine kidney epithelial cells, and mink lung cells. It is concluded that industrial whey extracted in this form constitutes an important source of potent growth-promoting agents for the supplementation of mesodermal-derived cell cultures.

Etanercept decreases tumor necrosis factor-α activity in chronic wound fluid

High levels of tumor necrosis factor‐α (TNF‐α), a pro‐inflammatory cytokine, are present in the wound fluid of chronic nonhealing wounds. This leads to increased inflammation, cytokine expression, and ultimately results in impaired wound healing and tissue destruction. Etanercept is a recombinant fusion protein that consists of the soluble TNF receptor (p75) linked to the Fc portion of human IgG1. It is an effective inhibitor of TNF‐α and has been shown to provide rapid and sustained improvement in rheumatoid arthritis by acting as a soluble receptor binding TNF‐α and preventing its proinflammatory activities. Therefore, the aim of this study was to determine whether Etanercept could inhibit TNF‐α activity in chronic wound fluid. Wound fluid was collected from the venous leg ulcers of 16 different patients. The effect of Etanercept on TNF‐α activity was evaluated using both a TNF‐α bioassay and an enzyme‐linked immunoassay. Etanercept was found to reduce the cytotoxic effect of chronic wound fluid on L929 fibroblasts by approximately 30% and neutralized TNF‐α binding in the enzyme‐linked immunoassay by up to 80%. Direct application of Etanercept to chronic wounds may therefore reduce the inflammatory activity of TNF‐α, which could reduce the chronicity of venous leg ulcers and thus aid in the healing of these wounds.

Identification of fibroblast growth factors in bovine cheese whey.

Acidic and basic fibroblast growth factors (FGF) were identified in bovine cheese whey after partial purification using a two step procedure. Cation-exchange chromatography produced a mitogen-rich extract which was loaded on to a heparin-sepharose column and eluted stepwise with 0.8, 1.2 and 2.0 M-NH4HCO3. Mitogenic activity was found in all three fractions by cell growth assays using Balb/c-3T3 fibroblasts. Immunoblotting identified acidic FGF in the 1.2 M-eluate and basic FGF in the 2.0 M-eluate, but neither acidic nor basic FGF was detected in the 0.8 M-fraction. Quantitative radioreceptor assays indicated 5.8 ng of acidic FGF-like activity and 19.8 ng of basic FGF-like activity per 1 whey in the appropriate eluates. This study represents the first direct demonstration of FGF in milk.

Insulin-like growth factor binding proteins (IGF-BPs) in bovine articular and ovine growth-plate chondrocyte cultures: their regulation by IGFs and modulation of proteoglycan synthesis

Cultured chondrocytes respond to insulin-like growth factors (IGFs) by increasing the production of proteoglycans and insulin-like growth factor binding proteins (IGF-BPs). To investigate the biological effects of IGFs and IGF-BPs, isolated bovine articular and ovine growth-plate chondrocytes were cultured at high density in the presence of IGF-1, and its truncated form, des (1-3) IGF-I. Both growth factors stimulated the production of IGF-BPs in articular and growth-plate chondrocyte monolayers. Western ligand blots showed that bovine articular chondrocytes released two forms of IGF-BPs into conditioned medium with molecular weights of 29 and 31 kDa. Ovine growth-plate chondrocytes released four different forms of IGF-BPs of approx. 22, 24; 29-30 and 34 kDa. IGF-I and des (1-3) IGF-I stimulated total proteoglycan synthesis by articular chondrocytes up to 1.5-fold. The truncated analogue was more potent at lower concentrations, particularly in stimulating incorporation of newly synthesized proteoglycans into the cell-layer. The maximal stimulation of proteoglycan synthesis in ovine growth-plate chondrocyte culture was 3-fold with des (1-3) IGF-I, while IGF-I enhanced proteoglycan production by only 2-fold over the concentrations used. Our results suggest that endogenous IGF-BPs in chondrocyte cultures act as a part of a feed-back mechanism which diminishes the bioactivity of IGF-I.

Paracellular transport of insulin‐like growth factor‐I (IGF‐I) across human umbilical vein endothelial cell monolayers

Insulin‐like growth factors (IGFs) are well defined mitogens and growth promoters, which are found in blood associated with high affinity IGF binding proteins (IGFBPs). In vivo, the endothelium is potentially the primary site of uptake of IGFs or IGF‐IGFBP complexes from blood for transport to the extravascular space. However, the pathway and mechanisms by which IGFs cross the endothelial cell barrier are not known. The presence of high affinity receptors for IGF‐I and IGF‐II on human umbilical vein endothelial (HUVE) cells was demonstrated by (i) radio‐receptor assays using both IGF‐I and IGF‐II and (ii) affinity label cross‐linking studies. In addition, Western ligand blotting and immunoblotting revealed that IGFBP‐2, ‐3, and ‐4 are secreted into serum‐free media conditioned by confluent HUVE cell monolayers. To study transendothelial migration of IGF‐I, HUVE cells were grown on microporous membranes in a bichamber system. When compared with membranes without cells, HUVE monolayers restricted the passage of 125I‐IGF‐I and [3H]inulin, whereas the control Madin Darby canine kidney (MDCK) cell line virtually excluded all passage of these molecules. Transport of 125I‐IGF‐I across HUVE cell monolayers was not significantly different to that of [3H]inulin, a paracellular probe. Moreover, 125I‐IGF‐I transport was not inhibited by either excess unlabelled IGF‐I or a monoclonal antibody to the type I IGF receptor at a concentration shown to inhibit 125I‐IGF‐I binding to HUVE cell monolayers. Our findings show that the movement of free IGF‐I across HUVE cell monolayers occurs via a paracellular route and not by a receptor‐mediated, transcellular pathway. J. Cell. Physiol. 170:290–298, 1997.

Mitogenic bovine whey extract modulates matrix metalloproteinase‐2, ‐9, and tissue inhibitor of matrix metalloproteinase‐2 levels in chronic leg ulcers

Matrix metalloproteinases (MMPs) and their tissue inhibitors play important roles in the wound‐healing process. An imbalance in the expression of these molecules is thought to contribute to the failure of chronic ulcers to heal. We investigated whether a mitogenic bovine whey extract enriched with growth factors modulated the expression and activity of MMP‐2 and ‐9, and the tissue inhibitor of MMP‐2 (TIMP‐2) in chronic leg ulcers. Wound fluids and biopsies were collected from chronic leg ulcer patients whose ulcers were treated topically for 4 weeks with placebo or mitogenic bovine whey extract at concentrations of 2.5, 10, and 20 mg/mL. The levels of MMP‐2 and ‐9 in wound fluid samples was assessed by gelatin zymography and showed a decrease in active MMP‐2 in the 2.5 and 10.0 mg/mL mitogenic bovine whey extract‐treated ulcers compared with placebo (p<0.05). Immunohistochemical analysis of ulcer biopsies for MMP‐2, ‐9, and TIMP‐2 expression showed a reduction in the number of MMP‐2–positive dermal fibroblasts in the mitogenic bovine whey extract‐treated ulcers compared with pretreatment biopsies (p<0.05) that persisted over the course of the study. In contrast, a transient increase in the number of MMP‐9– and TIMP‐2–positive cells was observed in mitogenic bovine whey extract treated ulcer biopsies compared with pretreatment levels (p<0.05). These results show that topical application of mitogenic bovine whey extract was able to modulate the expression of MMP‐2, ‐9, and TIMP‐2 in chronic leg ulcers and that its constituent growth factors may have the potential to redress the proteolytic imbalance observed in nonhealing chronic ulcers.

Clearance of IGFs and insulin from wounds: effect of IGF-binding protein interactions

We have examined the role binding proteins have in regulating the clearance of exogenous growth factors from wounds. Hunt-Schilling chambers were subcutaneously implanted in rats, and the clearance of insulin-like growth factor (IGF) I from the chamber wound fluid was compared with IGF-II, LR3-IGF-I, which binds poorly to IGF-binding proteins (IGFBP), or insulin. Elimination rate constants of the slow phase of the decay curves did not differ between IGF-I and IGF-II. However, LR3-IGF-I and insulin were cleared more rapidly from wound fluid than IGF-I so that the half-lives for IGF-I, IGF-II, LR3-IGF-I, and insulin were 872, 861, 563, and 324 min, respectively. In wound fluid, minimal degradation of the IGFs occurred, whereas insulin was degraded considerably. The increased clearance of LR3-IGF-I and insulin equated with a reduced association with wound fluid IGFBPs, and increased amounts of radioactivity of these peptides were detected in the circulation and urine. These results show that this model of wound repair may be of use in examining the kinetics of growth factors and other bioactive molecules in extravascular spaces and support the hypothesis that IGFBPs can be significant regulators of IGF bioavailability in vivo.We have examined the role binding proteins have in regulating the clearance of exogenous growth factors from wounds. Hunt-Schilling chambers were subcutaneously implanted in rats, and the clearance of insulin-like growth factor (IGF) I from the chamber wound fluid was compared with IGF-II, LR3-IGF-I, which binds poorly to IGF-binding proteins (IGFBP), or insulin. Elimination rate constants of the slow phase of the decay curves did not differ between IGF-I and IGF-II. However, LR3-IGF-I and insulin were cleared more rapidly from wound fluid than IGF-I so that the half-lives for IGF-I, IGF-II, LR3-IGF-I, and insulin were 872, 861, 563, and 324 min, respectively. In wound fluid, minimal degradation of the IGFs occurred, whereas insulin was degraded considerably. The increased clearance of LR3-IGF-I and insulin equated with a reduced association with wound fluid IGFBPs, and increased amounts of radioactivity of these peptides were detected in the circulation and urine. These results show that this model of wound repair may be of use in examining the kinetics of growth factors and other bioactive molecules in extravascular spaces and support the hypothesis that IGFBPs can be significant regulators of IGF bioavailability in vivo.

New therapeutics from a dairy byproduct—Cheese whey

An efficient process has been developed for extraction of growth factors, antimicrobial agents, and minor whey proteins from bovine cheese whey. The whey‐derived extract stimulates growth of mesodermal cell lines in culture but inhibits proliferation of epithelial‐derived cells. Known growth factors characterised in the material do not account for its complete biological activity in cultured fibroblasts, suggesting the presence of unidentified cell mitogens. The protein extract is being investigated for use in prevention and repair of gastrointestinal injury and surface wounds. In preclinical studies with experimental animal models, the protein extract has been shown to reduce damage in the small bowel and oral mucosa associated with ablative cytotoxic drug regimens, as well as to promote healing of incisional and excisional surface wounds. Human trials of the whey extract incorporating novel protein delivery systems are now under way to assess its safety and efficacy. If successful, the whey‐based extract will offer a valuable source of natural cell mitogens with widespread clinical application in preventing tissue damage and stimulating wound repair. Drug Dev. Res. 46:286–291, 1999.

Growth hormone but not insulin-like growth factor-I improves wound strength in pigs.

Systemic growth hormone and locally administered insulin‐like growth factor‐I have been shown in a number of studies to improve the breaking strength of incisional wounds, especially in compromised animals. The objective of the present study was to compare these two agents when administered subcutaneously distant from an incisional wound site in pigs, as well as to examine effects of a combination growth hormone/insulin‐like growth factor treatment. Growth hormone was shown to increase wound breaking strength in two experiments, whereas insulin‐like growth factor‐I or a more potent analog had no effect. Moreover, breaking strength was only minimally improved above the vehicle groups by the combination of growth hormone and insulin‐like growth factor‐I. These effects could not be explained by changes in plasma insulin‐like growth factor‐I concentrations which were highest in the combination groups, nor by plasma insulin‐like growth factor binding protein‐3 which was raised equally whenever growth hormone was administered. We conclude that systemic growth hormone but not insulin‐like growth factor‐I improves wound strength in normal pigs, whereas insulin‐like growth factor‐I reduces the magnitude of the growth hormone effect by an unknown mechanism.

Identification of a novel FcγRIII receptor that is up‐regulated in fetal wound healing

The mid‐gestation fetus is able to heal skin wounds rapidly and without scarring, an ability that is lost as development proceeds. The aim of this study was to identify novel genes involved in this process. We established an ex vivo wound model from embryonic rats and showed that over 72 hours, embryonic day 17 wounds reepithelialized and closed whereas day 19 wounds did not. To investigate the molecular basis of this phenomenon we analyzed changes in gene expression using differential display polymerase chain reaction. We characterized one transcript that was strongly up‐regulated in the healing response of wounded, day 17 skin. It encodes a protein of 249 amino acids with striking similarity to the human low‐affinity receptor for the Fc portion of IgG (FcγRIII), suggesting that it is a novel member of the FcγR family, which we named FcγRIII‐X. A wound‐healing timecourse shows that FcγRIII‐X was up‐regulated in healing, wounded day 17 skin but not in nonhealing, wounded day 19 skin and that its up‐regulation was accelerated in skin with multiple wounds. We suggest that up‐regulation of FcγRIII‐X may contribute to scarless healing of fetal skin.