Contribution of circulating IGF-I to wound repair in GH-treated rats.

Systemic administration of growth hormone (GH) stimulates granulation tissue formation, increases collagen deposition, improves the breaking strength of incisional wounds, and decreases donor-site healing times in burn patients. The possible role for circulating IGF-I in mediating this effect of growth hormone has not been investigated. To assess the relative effects of systemic IGF-I and GH on dermal repair, incisional wounds were created on the backs of male Sprague-Dawley rats treated with GH, or IGF-I or a combination of GH and IGF-I. After 7 days of treatment, wound strips were taken for wound strength and immunohistochemical analysis. Uninjured skin and liver samples were collected for mRNA analysis and plasma samples were taken at the completion of the experiment to determine circulating IGF-I levels. Increased circulating IGF-I levels and increased weight gain were observed only in the IGF-I and IGF-I+GH treatment groups, although steady-state igf-I levels were not altered in liver and uninjured skin after 7 days in any treatment group. IGF-I treatment had no positive effects on wound repair. Wound strength was increased with GH treatment only and associated with an increase in the intensity of IGF-I immunostaining in the granulation tissue of GH-treated animals. In line with the wound strength data, co-administration of IGF-I resulted in the decreased intensity of IGF-I immunostaining. We conclude that the GH-stimulated increase in wound strength is not mediated via endocrine-derived IGF-I and that only locally produced IGF-I acting in an autocrine or paracrine fashion contributes to the regulation of the wound repair process.

Hepatocyte growth factor and macrophage-stimulating protein are upregulated during excisional wound repair in rats.

Hepatocyte growth factor (HGF) and macrophage-stimulating protein (MSP) are structurally related molecules that stimulate epithelial cell proliferation and migration. MSP also acts directly as a chemoattractant for resident macrophages. These activities are integral to the wound repair processes of inflammation, epithelialization and tissue remodelling. To begin to examine the involvement of HGF and MSP in healing of cutaneous wounds we have mapped the temporal expression of these two molecules and their receptors, MET and RON respectively, in adult rat excisional wounds. Four 2x2-cm full-thickness excisional wounds were created on the dorsum of 18 rats, and biopsies were taken through the wounds at 3, 5, 7, 14, 21, and 28 days postwounding. These biopsies were analyzed using immunofluorescent staining and in situ hybridization (ISH). The number of cells staining positively for HGF and MET significantly increased in response to wounding. HGF staining and mRNA peaked at 7 days postwounding whereas MET was upregulated earlier, peaking after 3 days. Both HGF and MET protein were observed in fibroblasts of the dermis and in the newly forming granulation tissue. ISH studies also revealed that fibroblasts at the wound edges and within the newly forming granulation tissue also expressed HGF and c-met mRNA. Immunofluorescent staining revealed both MSP and RON within the wound, with maximum staining occurring between 7 and 21 days for both the ligand and receptor. In addition, MSP co-localized with a small subset of ED1-positive cells (monocytes). In contrast, ED2-positive cells (macrophages) did not co-localize with MSP. Thus, increased expression of HGF, MSP and their receptors MET and RON respectively was observed in response to wounding. Furthermore, MSP co-localization with a subset of monocytes may confirm a role for MSP in the activation of mature macrophages, which may be important in tissue remodelling.

Effect of healing on the expression of transforming growth factor beta(s) and their receptors in chronic venous leg ulcers.

The transforming growth factor betas are of major importance in the wound repair process; however, no studies to date have investigated the role of the transforming growth factor beta receptors in chronic venous leg ulcers or what effect healing has on these proteins. To determine whether the transforming growth factor beta peptides and their receptors are expressed in chronic venous wounds, we used immunofluorescent analysis and quantitative competitive reverse transcription polymerase chain reaction to identify the protein and mRNA expression, respectively. Biopsy samples from wounds and normal skin were collected from 12 patients with chronic venous leg ulcers and three patients undergoing reconstructive surgery, respectively. Additionally four of the chronic venous leg ulcer patients were re-biopsied between 2 and 8 wk after the first biopsy when the wounds had entered the healing phase. The tissue excised from the ulcers included the surrounding intact skin, the ulcer edge, and the ulcer base. Immunofluorescent staining for transforming growth factors beta1, beta2, and beta3 was observed within the epidermis of the skin surrounding the chronic venous ulcers and in fibroblasts and inflammatory cells of the dermis, although this staining was not as strong as that seen in normal unwounded skin. Very little staining could be seen within the ulcers for any of the ligands, however. In contrast the transforming growth factor beta type I receptor was observed throughout the ulcers and the normal unwounded skin biopsies, particularly in the basal epidermal cells. No immunofluorescence for the type II transforming growth factor beta receptor was observed in any of the ulcer biopsies investigated, although it was observed throughout the epidermis and in fibroblasts and inflammatory cells in the surrounding skin. Quantitative, competitive reverse transcription polymerase chain reaction was used to analyze mRNA expression for transforming growth factor beta1 and the type II receptor in the nonhealing ulcers and normal unwounded skin biopsies. These studies revealed that transforming growth factor beta1 and transforming growth factor beta receptor II mRNA was expressed in all the chronic nonhealing ulcers albeit at very low levels for the type II receptor. In marked contrast to the staining observed in nonhealing chronic ulcers, positive immunostaining was observed for the transforming growth factor betas and both the type I and type II receptors in healing ulcers. These results suggest that the absence of a viable receptor complex for the transforming growth factor betas in nonhealing chronic venous ulcers may contribute to wound chronicity.

Mitogenic whey extract stimulates wound repair activity in vitro and promotes healing of rat incisional wounds.

The ability of single growth factors to promote healing of normal and compromised wounds has been well described, but wound healing is a process requiring the coordinated action of multiple growth factors. Only the synergistic effect on wound healing of combinations containing at most two individual growth factors has been reported. We sought to assess the ability of a novel milk-derived growth factor-enriched preparation ¿mitogenic bovine whey extract (MBWE), which contains six known growth factors, to promote repair processes in organotypic in vitro models and incisional wounds in vivo. MBWE stimulated the contraction of fibroblast-populated collagen lattices in a dose-dependent fashion and promoted the closure of excisional wounds in embryonic day 17 fetal rat skin. Application of MBWE increased incisional wound strength in normal animals on days 3, 5, 7, and 10 and reversed the decrease in wound strength observed following steroid treatment. Wound histology showed increased fibroblast numbers in wounds from normal and steroid-compromised animals. These data suggest the mixture of factors present in bovine milk exerts a direct action on the cells of cutaneous wound repair to enhance both normal and compromised healing.

Transport of circulating IGF-I and LR3IGF-I from blood to extracellular wound fluid sites in rats.

Significant levels of IGF-I are found in wound fluid. The contribution that systemic IGF-I makes to the total IGF-I pool in wounds and the influence of IGF binding proteins (IGFBPs) on the delivery of systemic IGF-I to these wound sites has not been established. In the present series of experiments, we have shown that IGF-I flux across a model endothelial cell barrier is decreased in the presence of IGFBPs, whereas flux of Long R(3)IGF-I (LR(3)IGF-I, an IGF-I analogue with low affinity for IGFBPs) is unaffected. On the basis of these findings, the transport of IGF-I and LR(3)IGF-I from blood to extracellular wound fluid was assessed. Wound chambers were implanted subcutaneously in the backs of adult male rats and left in place for 14 days. A single i.v. bolus of either (125)I-IGF-I or (125)I-LR(3)IGF-I (10x10(6) c.p.m.) was administered via a jugular catheter and wound fluid and plasma samples taken at sequential time points between 5 and 240 min. (125)I-LR(3)IGF-I was removed from the circulation more rapidly than (125)I-IGF-I in both sham control and chamber implanted rats. Although implantation of the chambers did not alter the pharmacokinetic parameters of (125)I-IGF-I, significant increases in the steady state volume of distribution, clearance rate and half-life were recorded for (125)I-LR(3)IGF-I. In addition, significantly more intact (125)I-LR(3)IGF-I was recovered in wound fluid than (125)I-IGF-I at each time point, although only 0.08% of administered (125)I-LR(3)IGF-I was recovered per ml of wound fluid at 240 min. Compared with plasma, a greater proportion of wound fluid IGF-I radioactivity had distributed to the lower molecular weight IGFBPs or existed as free peptide. However, a small amount of wound fluid (125)I-IGF-I was detected in the 150 kDa region 30 min after injection. A greater proportion of (125)I-LR(3)IGF-I was associated with the lower molecular weight IGFBPs or existed as free peptide in both wound fluid and plasma. These data point to the importance of IGFBPs in determining the pharmacokinetic parameters of IGF-I in an extracellular fluid-expanded state. They also suggest only a minor role for endocrine IGF-I in surface wound repair.

Identification of betacellulin as a major peptide growth factor in milk: purification, characterization and molecular cloning of bovine betacellulin.

Betacellulin (BTC), a member of the epidermal growth factor (EGF) family of peptide growth factors, was purified from a growth-factor-enriched whey fraction of bovine milk by a combination of ion-exchange chromatography, gel-filtration chromatography, affinity chromatography and reverse-phase HPLC. Bovine BTC (bBTC) had an apparent molecular mass of 21-22 kDa on SDS/PAGE and exists in a glycosylated form. The cDNA encoding bBTC was obtained by a combination of 5' and 3' rapid amplification of cDNA ends ('RACE'). The primary translation product consists of 178 amino acid residues containing a putative signal sequence, a transmembrane domain, the mature BTC domain and a cytoplasmic domain containing a highly hydrophilic Arg-Lys-rich region similar to that of mouse BTC and human BTC. The amino acid sequence of the bBTC precursor was 88% identical with human BTC and 79% identical with mouse BTC. The bBTC gene was found to be expressed in a wide range of tissues, including the mammary gland. The identification of BTC in milk raises the possibility that it has a major role in the growth and development of the neonatal gastrointestinal tract.

Transport of IGF-I across epithelial cell monolayers.

Epithelial cells line the lumens of organs including the gastrointestinal tract, kidney tubules and respiratory airways, where they regulate the transport of electrolytes and the movement of macromolecules. The current study aimed to investigate the transport of IGF-I across epithelial cell barriers. Epithelial cell lines derived from gut (IEC-6), kidney (MDBK) and lung (Mv1Lu) were shown to possess high-affinity, functional receptors for IGF-I and formed tight junctions in monolayer culture. To investigate the transport of IGF-I, the three cell lines were grown on microporous filters in a bi-chamber system. In comparison with filters without cells, IEC-6 and Mv1Lu epithelial cell monolayers restricted the passage of (125)I-IGF-I and [(3)H]inulin, whereas the MDBK cells virtually occluded any passage of these molecules. Transport of (125)I-IGF-I across the epithelial cell monolayers was significantly less than that of [(3)H]inulin, suggesting that the binding of (125)I-IGF-I to high-affinity IGF receptors or IGF-binding proteins retarded its transport. Moreover, (125)I-IGF-I transport was not inhibited by the presence of excess unlabelled IGF-I. Our findings provide evidence for the restricted diffusion of intact, free IGF-I across gut, kidney and lung epithelial cell monolayers via a paracellular or low-affinity transcellular pathway.

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.

Insulin-like growth factor binding protein-5 proteolytic activity in ovine articular chondrocyte culture.

We have previously demonstrated that ovine articular chondrocytes synthesise and release insulin-like growth factor binding protein-5 (IGFBP-5) which subsequently undergoes proteolysis in the tissue culture medium. The IGFBP-5 proteolytic activity has now been characterised and its substrate specificity analysed using recombinant IGFBP-5 and purified chondrocyte-derived IGFBPs. Iodinated human recombinant IGFBP-5 was incubated with chondrocyte culture or conditioned medium in the presence or absence of various inhibitors. Serine protease inhibitors aprotinin and heparin effectively inhibited the breakdown of IGFBP-5. Furthermore, insulin-like growth factor-I (IGF-I) but not its structural analogues with reduced affinity for IGFBP-5, was also able to partially protect IGFBP-5 from degradation indicating that the association of IGF with the binding protein was required for the inhibition of the proteolytic activity. The inflammatory cytokine interleukin-1 did not have any effect on IGFBP-5 proteolysis. The proteolytic activity appears to be IGFBP-5-specific since the incubation of chondrocyte-derived IGFBPs with chondrocyte conditioned medium resulted in the loss of IGFBP-5 while the levels of the other two IGFBPs (IGFBP-2 and a 24 kDa IGFBP) remained unchanged. In conclusion, we show that IGFBP-5 is specifically cleaved by a serine protease released by primary cultures of ovine articular chondrocytes and also demonstrate the ability of IGF-I to inhibit the proteolytic activity both in cell culture and in cell-free conditions.

Regulation of insulin-like growth factor-binding protein-5 by insulin-like growth factor I and interleukin-1alpha in ovine articular chondrocytes.

Insulin-like growth factors (IGFs) contribute to the maintenance of the cartilage matrix by stimulating proteoglycan synthesis. In contrast, interleukin-1 (IL-1), an inflammatory cytokine, suppresses the synthesis of proteoglycans. In pathological conditions the chondrocytes' responsiveness to IGF-I is decreased, and elevated levels of IGF-binding proteins (IGFBPs) have been implicated as a possible cause. The aim of this study was to investigate the effects of IGF-I and IL-1 on IGFBP production by ovine articular chondrocytes (OAC) and the roles of these IGFBPs in the regulation of proteoglycan synthesis. As revealed by Western ligand and immunoblotting, OACs secreted IGFBP-2 and a 24-kDa IGFBP in culture medium under basal conditions. Exposure of the cells to IGF-I for 48 h resulted in the appearance of IGFBP-5 in the medium. Des(1-3)IGF-I, an IGF-I analog with reduced affinity for IGFBPs, also increased the level of IGFBP-5, but to a lesser extent than IGF-I, whereas LR3IGF-I, which has virtually no affinity for IGFBPs, had no effect on IGFBP-5. Furthermore, IGFBP-5 underwent a time-dependent limited proteolysis when incubated with OAC-conditioned medium, degrading into 22- and 16-kDa fragments. The degradation of IGFBP-5 was significantly inhibited by IGF-I, but not by des(1-3)IGF-I or LR3IGF-I. Basic fibroblast growth factor, transforming growth factor-beta, and platelet-derived growth factor had no effect on OAC IGFBPs. However, IL-1alpha increased the IGFBP-5 level in a dose-dependent manner, showing maximum activity at 200 U/ml. Furthermore, IL-1alpha, but not IGF-I, induced IGFBP-5 messenger RNA expression, as assessed by Northern blot analysis. Coincubation of IGF-I with IL-1alpha resulted in a substantially increased IGFBP-5 protein level, suggesting a synergism between the mechanisms of action of these two factors. Des(1-3)IGF-I and LR3IGF-I were 10 times more potent than IGF-I in stimulating proteoglycan synthesis, indicating inhibition of IGF-I activity by endogenous IGFBPs. IL-1alpha reduced the IGF-I bioactivity, but had no effect on the activities of the IGF-I analogs, thus implying that locally produced IGFBPs, particularly IGFBP-5, which was substantially increased when IGF-I and IL-1alpha were coincubated, mediated the reduction of the IGF-I activity. Our results demonstrate that IGF-I and IL-1alpha synergistically increase the level of IGFBP-5 in OAC by inhibiting the proteolysis and stimulating the expression of IGFBP-5, respectively. Furthermore, the attenuation of IGF-I-stimulated proteoglycan synthesis by IL-1alpha in OAC appears to be mediated by chondrocyte IGFBPs. We conclude that locally produced IGFBPs, in particular IGFBP-5, may play a critical role in the regulation of cartilage matrix degradation in inflammatory and degenerative arthritides.

The mechanism of excisional fetal wound repair in vitro is responsive to growth factors.

We have investigated the ability of fetal rat skin to heal an excisional wound in vitro. Skin from the backs of E17-E19 rats was wounded using a 1-mm diameter cutting needle and suspended in culture on a 6-pin cradle for 72 h. Neither contraction nor epithelial closure was observed within wounds created in skin from E19 embryos. In contrast, wounds in E17 skin contracted to 35-50% of their original area over 72 h, although, in the absence of serum, complete wound closure was not observed. Addition of FBS at the time of culture resulted in the movement of the epithelium over the dermal margins of the wound to effect complete closure. Histological sections through these healed wounds revealed an epithelial bridge spanning the dermal margins of the wound. A similar mechanism of repair was observed in the presence of day 14 adult wound fluid. The response of wounds in E17 skin to a range of growth factors was then assessed in an attempt to reproduce the serum response under defined conditions. Insulin-like growth factor I or epidermal growth factor did not significantly affect wound closure. Basic fibroblast growth factor, transforming growth factor-beta, or platelet-derived growth factor did promote wound closure although, in contrast to the serum-induced response, wound histology revealed repair had been achieved by dermal fibroblasts that occupied the space between the epithelial margins of the healed wound. We have therefore shown that the epithelial component of fetal wound repair proceeds in organ-cultured fetal skin in the absence of an adhesive substrate over which to migrate and is dependent on the source of trophic factors. The inability of skin taken from the E19 embryo to heal in vitro suggests a developmental switch in the mechanism of wound epithelialization.

Platelet-derived growth factor, insulin-like growth factors, fibroblast growth factors and transforming growth factor beta do not account for the cell growth activity present in bovine milk.

Cation-exchange chromatography effectively concentrates the cell growth activity present in whey and we have used this process as a basis to characterise further the growth factors present in bovine milk. Under neutral conditions, total bioactivity in the growth factor-enriched cation-exchange fraction chromatographed with an apparent molecular mass of 80-100 kDa. In contrast, acid gel-filtration chromatography resolved two peaks of cell growth activity. A peak at 15-25 kDa contained the bulk of growth activity for Balb/c 3T3 fibroblasts while bio-activity for L6 myoblasts and skin fibroblasts eluted with a molecular mass of 6 kDa. A peak of inhibitory activity for Mv1Lu and MDCK cells also eluted at 15-25 kDa. Both IGF-I and IGF-II were purified from fractions that eluted at 6 kDa, although the IGF peptides alone did not account for the total bioactivity recovered. Platelet-derived growth factor (PDGF), identified by radioreceptor assay, eluted at a slightly higher molecular mass than the peak of growth activity for Balb/c 3T3 cells, and an anti-PDGF antibody was without effect on the growth of Balb/c 3T3 cells in response to the whey-derived factors. Further purification of the inhibitory activity for epithelial cells yielded a sequence for transforming growth factor beta (TGF-beta), and all inhibitory activity for Mv1Lu cells was immunoneutralised by an antibody against TGF-beta. In contrast, this antibody decreased the growth of Balb/c 3T3 fibroblasts in the whey-derived extract by only 10%. Finally, a cocktail of recombinant growth factors containing IGF-I, IGF-II, PDGF, TGF-beta and fibroblast growth factor 2 stimulated growth of Balb/c 3T3 cells to a level equivalent to only 51% of that observed in the milk-derived growth factor preparation. We conclude that: (i) cell growth activity recovered from bovine whey is present in acid-labile high molecular weight complexes; (ii) all cell growth inhibitory activity for epithelial cells can be accounted for by TGF-beta; (iii) IGF-I and IGF-II co-elute with the major peak of activity for L6 myoblasts and skin fibroblasts, although the IGF peptides alone do not explain the growth of these cells in the whey-derived extract; and (iv) neither PDGF nor TGF-beta account for the 15-25 kDa peak of Balb/c 3T3 growth activity. These data suggest the presence of additional mitogenic factors in bovine milk.

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.

Transforming growth factor beta in bovine milk: concentration, stability and molecular mass forms.

Transforming growth factor beta (TGF-beta) is one of the predominant growth factors present in milk. The concentration, molecular mass forms and stability of TGF-beta in bovine milk were investigated using a standard bioassay measuring the growth inhibition of a milk lung epithelial cell line. Most of the TGF-beta bioactivity in milk was found to be in a latent form, which was also retained in the whey fraction. After acid activation, the total TGF-beta concentration was 4.3 +/- 0.8 ng and 3.7 +/- 0.7 ng TGF-beta per ml of milk and cheese whey respectively. Cation-exchange chromatography at pH 6.5 was used to concentrate latent whey-derived TGF-beta, which could be activated by transient exposure to extremes of pH, urea or heat. Heparin did not significantly activate milk-derived TGF-beta. Neutral gel filtration of the cationic whey fraction revealed a major peak of latent TGF-beta with a molecular mass of 80 kDa and a smaller peak at 600 kDa. Transient acidification of the cationic whey fraction prior to neutral gel filtration, or gel filtration under acidic conditions, released low molecular mass TGF-beta from both high molecular mass peaks. Whey-derived TGF-beta was purified using a five-step chromatographic procedure. An N-terminal sequence was obtained for TGF-beta 2, which accounted for over 85% of the TGF-beta bioactivity in whey. All TGF-beta activity in whey could be neutralised by a monoclonal antibody directed against TGF-beta 1, -beta 2 and -beta 3. The results suggest that the majority of TGF-beta in bovine milk is present in a small latent complex.

Insulin-like growth factor I (IGF-I) and IGF-Binding proteins in rat wound fluid.

Insulin-like growth factors (IGFs) play an important role in tissue repair, including healing of dermal and epidermal injury. In this study we have measured changes in the IGF:IGF-binding protein (IGFBP) profile of rat wound fluid (WF) collected after sc implantation of Hunt-Schilling chambers for 21 days. WF IGF-I levels 1 day after implantation were equivalent to plasma levels, then fell during the first 7 days before recovering to approximately two thirds of plasma levels by day 21. Western ligand blots of whole WF revealed a profile qualitatively similar to that found in plasma, although the intensity of the IGFBP-3 band was significantly less than that in plasma. Neutral gel chromatography of pooled day 14 WF, after in vitro incubation with [125I]IGF-I, separated the radioligand into three distinct regions of 150, 40, and 7.5 kDa. However, compared to plasma recovery of[125I]IGF-I in the 150-kDa region in WF was reduced, and that in the 40-kDa region was increased. Ligand blotting of the WF-derived neutral gel fractions revealed IGFBP-3 within the 150-kDa complex. Incubation of WF with plasma (1:1, vol/vol) resulted in a progressive decline in the intensity of the plasma IGFBP-3 band. Protease inhibitors, including EDTA, antipain, or aprotonin, inhibited this process. We have described the changes over time in WF IGF-I concentrations, characterized the IGFBP profile, and demonstrated the presence of IGFBP-3 proteolytic activity in WF. The latter may play a role in the regulation of IGF bioavailability during the repair process.

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

We 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.

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.

Investigation of the ability of several naturally occurring and synthetic polyanions to bind to and potentiate the biological activity of acidic fibroblast growth factor.

The ability of several animal, plant, and bacterial derived polyanions (PAs) as well as synthetic PAs to compete with heparin for the binding of acidic fibroblast growth factor (aFGF) was correlated with their ability to potentiate the mitogenic and neurotrophic actions of this factor. Dextran sulphate, kappa-carrageenan, pentosan sulphate, polyanethole sulfonate, heparin, and fucoidin competed for the heparin binding site on aFGF at relatively low concentrations (< 50 micrograms/ml). lambda-carrageenan, iota-carrageenan, and polyvinyl sulphate exhibited lower affinity for aFGF, whereas hyaluronic acid, dermatan sulphate, chondroitin-6-sulphate, chondroitin-4-sulphate, and uncharged dextran displayed very low or no demonstrable affinity. Potentiation of the mitogenic action of aFGF for Balb/c 3T3 fibroblasts tended to be in general agreement with the aFGF binding affinity of the PAs. However, polyanethole sulfonate, the carrageenans, polyvinyl sulphate, fucoidin, and pentosan sulphate exerted a mitogenic action on the 3T3 cells that was independent of, and in addition to, the ability of these GAGs to potentiate the action of aFGF. The ability to potentiate the neurotrophic action of aFGF for E8 chick ciliary neurons was a general property of those PA with low or no activity in the mitogen assay. Thus hyaluronic acid, dermatan sulphate, chondroitin-4-sulphate, chondroitin-6-sulphate, and even unchanged dextran all potentiated aFGF induced neuronal survival. The differential effects of these PA in potentiating the biological activities of aFGF are discussed in relation to their ability to compete for the heparin-binding site of aFGF.

Ability of different chemically modified heparins to potentiate the biological activity of heparin-binding growth factor 1: lack of correlation with growth factor binding.

A range of chemically modified heparins was examined for their ability to bind heparin-binding growth factor 1 (HBGF-1; acidic fibroblast growth factor) and potentiate the in vitro mitogenic and neurotrophic activity of HBGF-1. It was found that carboxyl-reduced heparin bound HBGF-1 as effectively as the native heparin molecule. Totally desulfated heparin and N-desulfated heparin lack HBGF-1-binding capacity, and substitution of the exposed amino group with acetyl or acetoacetyl groups only partially restored binding capacity, indicating that N-sulfates only play a limited role in growth factor binding. However, the failure of totally desulfated, N-resulfated heparin to interact with HBGF-1 demonstrated that N-sulfates alone are insufficient and ester sulfates are absolutely essential for HBGF-1 binding. In contrast, the ability of the modified heparins to potentiate the mitogenic activity of HBGF-1 correlated only to a limited extent with their affinity for HBGF-1. Thus, the carboxyl-reduced molecule which displayed similar affinity for HBGF-1 as native heparin was consistently less potent in augmenting mitogenesis. Similarly, the N-acetylated and the N-acetoacetylated species, which had much lower affinity for HBGF-1 than the carboxyl-reduced molecule, conferred similar biological activity to HBGF-1 whereas N-desulfated heparin, which was unable to bind growth factor, potentiated the mitogenic activity of HBGF-1 for both 3T3 and HUVE cells. In contrast, the neurotrophic activity of HBGF-1 was potentiated by modified heparin species which failed to bind HBGF-1 and were without activity in the mitogenic assays.(ABSTRACT TRUNCATED AT 250 WORDS)