Selective pick-up of increased iron by deferoxamine-coupled cellulose abrogates the iron-driven induction of matrix-degrading metalloproteinase 1 and lipid peroxidation in human dermal fibroblasts in vitro: a new dressing concept.

Using atomic absorption spectrum analysis, we found iron levels in exudates from chronic wounds to be significantly increased (3.71 +/- 1.56 micromol per g protein) compared to wound fluids from acute wounds derived from blister fluids (1.15 +/- 0.62 micromol per g protein, p < 0.02), drainage fluids of acute wounds (0.87 +/- 0.34 micromol per g protein, p < 0.002), and pooled human plasma of 50 volunteers (0.42 micromol per g protein). Increased free iron and an increase in reactive oxygen species released from neutrophils represent pathogenic key steps that --via the Fenton reaction - are thought to be responsible for the persistent inflammation, increased connective tissue degradation, and lipid peroxidation contributing to the prooxidant hostile microenvironment of chronic venous leg ulcers. We herein designed a selective pick-up dressing for iron ions by covalently binding deferoxamine to cellulose. No leakage occurred following gamma sterilization of the dressing and, more importantly, the deferoxamine-coupled cellulose dressing retained its iron complexing properties sufficient to reduce iron levels found in chronic venous ulcers to levels comparable to those found in acute wounds. In order to study the functionality of the dressing, human dermal fibroblasts were exposed to a Fenton reaction mimicking combination of 220 microM Fe(III) citrate and 1 mM ascorbate resulting in a 4-fold induction of matrix-degrading metalloproteinase 1 as determined by a matrix-degrading metalloproteinase 1 specific enzyme-linked immunosorbent assay. This induction was completely suppressed by dissolved deferoxamine at a concentration of 220 microM or by an equimolar amount of deferoxamine immobilized to cellulose. In addition, the Fe(III) citrate and ascorbate driven Fenton reaction resulted in an 8-fold increase in malondialdehyde, the major product of lipid peroxidation, as determined by high pressure liquid chromatography. This increase in malondialdehyde levels could be significantly reduced in the presence of the selective pick-up dressing coupled with deferoxamine suggesting that the deferoxamine dressing, in fact, prevents the development of a damaging prooxidant microenvironment and also protects from unfavorable consequences like matrix-degrading metalloproteinase 1 and lipid peroxide induction.

Hydroactive dressings and serum proteins: an in vitro study.

An in vitro approach was used to obtain information about the compatibility of hydroactive dressing materials with the serum proteins which are thought to be relevant to wound healing. Artificial wound fluid was incubated with different hydroactive dressings (Cutinova hydro, Varihesive E, Comfeel Ulcer Dressing and Allevyn), and concentrations of total protein, albumin, immunoglobulin and growth factors were measured after one day of incubation. Cutinova hydro and Allevyn absorbed considerable amounts of fluid. Fluid uptake was lower for the hydrocolloid dressings. An unexpected finding was that Cutinova hydro showed an approximately two-fold increase over control values in the concentration of all proteins tested, indicating a selective absorption of water by this dressing. For the other dressings tested, zero or very low absorption of proteins was found, indicating a basically satisfactory protein compatibility.

Induction of beta 1 integrin synthesis by recombinant platelet-derived growth factor (PDGF-AB) correlates with an enhanced migratory response of human dermal fibroblasts to various extracellular matrix proteins.

Cell migration plays a major role during wound healing and is tightly controlled by a variety of growth factors and extracellular matrix proteins. The experiments reported here have been designed to study whether defined beta 1 integrins are involved in the platelet-derived growth-factor-AB (PDGF-AB)-modulated migratory response to collagen type I and to fibronectin. Preincubation of fibroblasts with PDGF-AB resulted in an up to 2.5-fold increase in the migratory response to collagen type I as well as fibronectin and to enhanced synthesis and cell surface expression of the alpha 2, alpha 3, alpha 5, and beta 1 integrin subunits. Function-blocking monoclonal antibodies against the common beta 1 integrin subunit dose-dependently inhibited the PDGF-AB-augmented migration of fibroblasts to collagen type I and fibronectin. The PDGF-AB-induced migration to collagen type I was also inhibited by antibodies against the alpha 2 integrin subunit, whereas the corresponding migration to fibronectin was almost completely blocked by the combined application of antibodies against the alpha 3 and the alpha 5 integrin subunits. Taken together, up-regulation of integrin synthesis and expression by human recombinant PDGF-AB correlate with an increase in the migratory response of dermal human fibroblasts to various extracellular matrix proteins and thus may contribute to an efficient regulation of cell migration during wound healing and tissue repair.

Localization of platelet-derived growth factor receptor subunit expression in chronic venous leg ulcers.

Cellular responses to platelet-derived growth factor, which affects all phases of the wound healing process, are dependent on the interaction of the growth factor with its cell surface receptors. Recently, we have shown that the platelet-derived growth factor-receptor was not expressed in uninjured human skin. In acute human wounds healing by secondary intention, both platelet-derived growth factor-receptor subunits were coordinately expressed, whereas no expression was found after reepithelialization at day 47. Even though impaired wound healing may be due to uncoordinated expression or the failure to express platelet-derived growth factor-receptor subunits, little is known regarding their expression in chronic ulcers. We studied the localization of platelet-derived growth factor-receptor expression in chronic venous leg ulcers of 15 patients with a median age of 73 years. Cryostat sections of biopsy specimens were immunostained with the use of antibodies against the alpha- and the beta-platelet-derived growth factor subunits. RNA was extracted from biopsy specimens and subjected to Northern blot analysis with the use of oligolabeled complementary DNA for the platelet-derived growth factor-receptor. Platelet-derived growth factor-receptor alpha- and beta-subunit expression was found in fibroblast-like cells within the wound bed and in cells beneath the epidermis of the wound edge. Platelet-derived growth factor-receptor beta-subunit expression was detected in endothelial cells of the vessels, in the granulation tissue, and the wound edge, whereas platelet-derived growth factor-receptor alpha-subunit was not expressed in endothelial cells of the uninjured skin. This finding suggests that the platelet-derived growth factor alpha-subunit may be involved in vessel formation during tissue repair. Both platelet-derived growth factor-receptor subunits were expressed at the messenger RNA level indicating that the synthesis is at least partly regulated at a pretranslational level. As the cellular responsiveness to growth factors depends on their specific receptors, our finding that both platelet-derived growth factor-receptor subunits are expressed in chronic venous ulcers substantiates the concept of therapeutic trials with recombinant platelet-derived growth factor.

Hyaluronan, heterogeneity, and healing: the effects of ultrapure hyaluronan of defined molecular size on the repair of full-thickness pig skin wounds.

The extracellular matrix macromolecule, hyaluronan, is thought to modulate wound healing. However, the molecular size of hyaluronan and contaminating associated proteins may be important determinants of these effects. We have examined the results of seven daily topical treatments of full-thickness skin wounds in pigs with ultrapure hyaluronan of defined molecular size. High molecular weight hyaluronan (>1000 kd) enhanced, whereas low molecular weight hyaluronan decreased, the rate of early wound contraction as compared with intermediate hyaluronan (molecular weight = 100 kd) and saline solution controls. Fracture strength at 21 days was reduced by high and intermediate molecular weight hyaluronan but not by low molecular weight hyaluronan. Wound perfusion, measured by means of a scanning laser-Doppler technique as a noninvasive indicator of angiogenesis, showed depression by high and intermediate molecular weight hyaluronan on day 3, but all forms of hyaluronan caused elevated blood flow on day 7. The architecture of granulation tissue in this wet healing model was highly organized, but no gross histologic differences were seen because of treatment. Different molecular species of hyaluronan have differential effects on contraction, angiogenesis, and the evolution of wound strength. Where hyaluronan is used as a treatment or vehicle for wounds, its precise composition should be specified.

Platelet-derived growth factor.

Platelet-derived growth factor (PDGF) is a potent activator for cells of mesenchymal origin. Two different PDGF chains termed A and B encoded by different genes have been identified leading to three different PDGF isoforms, the AA and BB homodimers and the AB heterodimer. All three forms have been observed in vivo and possess biological activity in vitro with the AA homodimer being the poorest cellular mitogen. The availability of highly purified recombinant PDGF isoforms was the initial basis for comparative studies in order to specify the different spectra of activity of the various PDGF species. This review is particularly focused on AB heterodimer as from the standpoint of heterologous gene expression, this species is the one with the highest demands concerning expression and purification protocols. This explains the fact that, in comparison to PDGF-BB, only very limited data on the in vivo application of PDGF-AB are available so far.

Wound therapy: growth factors as agents to promote healing.

The development of recombinant growth factors for the treatment of nonhealing or badly healing wounds has reached the clinical-trial phase. These first studies in humans are yielding valuable information on the physiological role of growth factors in different types of wounds, their mode of action and their stability under in vivo conditions. Data from these early trials in humans can be interpreted in the light of the continuing scientific progress being made in wound-healing research, and thus lead to an improvement in the design of future studies. Growth-factor-based therapeutics are expected to enter the marketplace in the middle of this decade, and to become a highly profitable sector of the health-care industry by the turn of the century.

Influence of a beta-adrenergic agonist on septic shock-induced alterations of phosphatidylcholine metabolism in rat lung.

Changes in surfactant function play an important part in the pathogenesis of adult respiratory distress syndrome (ARDS). Since beta-adrenergic agonists have been shown to exert a decisive influence on surfactant secretion, we studied the effect of fenoterol on lung phospholipid metabolism under conditions of experimental sepsis. Fenoterol administered to live rats increased the incorporation of choline into lung tissue by 80% in normal, by 35% in septic animals. It had no comparable effect on palmitate incorporation. It increased the activity of choline kinase in control animals, but had no additional effect on animals with increased values due to sepsis. Phosphotransferase activity diminished during sepsis was stimulated, and phospholipase activity reduced. Fenoterol restored phosphatidylcholine to normal levels in lung tissue and bronchoalveolar lavage and prevented lysophosphatidylcholine generation. Fenoterol also increased the amount of palmitate in phosphatidylcholine from bronchoalveolar lavage in septic animals. The results imply that a beta-adrenergic agonist influences the conditions of lung phospholipid metabolism altered by sepsis towards normal.