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For many years, lactate has been known to accelerate collagen deposition in cultured fibroblasts and, without detailed explanation, has been presumed to stimulate angiogenesis. Similarly, hypoxia has been linked to angiogenic effects and collagen deposition from cultured cells. Paradoxically, however, wound angiogenesis and collagen deposition are increased by breathing oxygen and decreased by hypoxia. Lactate accumulates to 4-12 mM in wounds for several reasons, only one of which is the result of hypoxia. Oxygen in wounds is usually low but can be increased by breathing oxygen (without change in lactate). We have reported that lactate elicits vascular endothelial growth factor (VECF) from macrophages, as well as collagen, some heat shock proteins, and VECF from endothelial cells, and collagen from fibroblasts, even in the presence of normal amounts of oxygen. Hypoxia exerts many of these same effects in cultured cells. In this study, we elevated extracellular lactate in wounds by implanting purified solid-state, hydrolysable polyglycolide. A steady-state 2-3 mM additional elevation of lactate resulted. With it, there was a significant short-term elevation of interleukin-1beta, a long-term elevation of VECF (2x) and transforming growth factor-beta1 (2-3x), a 50% elevation in collagen deposition, and a large reduction of insulin-like growth factor-1 (- 90%). We propose that lactate induces a biochemical "perception" of hypoxia and instigates several signals that activate growth factor/cytokine signals while the continued presence of molecular oxygen allows endothelial cells and fibroblasts to reproduce and deposit collagen. The data are consistent with ADP-ribosylation effects and oxidant signaling. (WOUND REP REG 2003;11:504-509)

Macrophages respond to various stimuli to produce angiogenic factors but few mechanistic details are known. We examined the effects of hypoxia, lactate and nicotinamide on the expression of vascular endothelial growth factor by cultured macrophages. These agents were chosen because they down-regulate polyadenosine diphosphoribose levels. Following exposure, conditioned media were analyzed for vascular endothelial growth factor protein. Nicotinamide adenine dinucleotide, polyadenosine diphosphoribose, and vascular endothelial growth factor mRNA were measured in the cellular fraction. Angiogenic capacity of the conditioned media was tested in rabbit corneas and Matrigel implants. All three agents, hypoxia, lactate and nicotinamide, elicited significantly increased levels of vascular endothelial growth factor mRNA and vascular endothelial growth factor in the conditioned media, and these levels were paralleled by their angiogenic activity. Polyadenosine diphosphoribose in the cellular fraction was correspondingly depressed. Anti-vascular endothelial growth factor antibody inhibited most of the angiogenic response whereas anti-basic fibroblast growth factor antibody had little effect. We propose that redox changes associated with the alteration of cellular nicotinamide adenine dinucleotide and polyadenosine diphosphoribose are involved in lactate-mediated VEGF expression.

Targeted disruption of the stromelysin-1 gene in mice causes a delay in excisional wound healing due to a failure in wound contraction. Therefore, we postulated that stromelysin-1 activity is responsible for initiating contraction. To test this hypothesis, we compared the contractile capacity of fibroblasts from stromelysin-1 knockout mice (strom-1 KO) with that of normal fibroblasts using a collagen gel contraction model. Fibroblast cultures were established from explants of skin and lung parenchyma from strom-1 KO and wild-type mice, then transferred to the surface of collagen gels. The extent of contraction was determined by measuring greatest gel diameter. Results demonstrated that (1) all fibroblasts contracted collagen gels in a uniform concentric fashion, (2) skin fibroblasts from both sets of mice exhibited greater gel contraction than did lung fibroblasts, and (3) strom-1 KO fibroblasts demonstrated significantly less contraction (21-23%) than wild-type fibroblasts. These data support the hypothesis that absence of stromelysin-1 results in defective fibroblast contraction that may contribute to delayed wound healing.

Complications of irradiated tissue include infections and impaired healing. Although fibrosis and hypovascularity contribute, a cellular mechanism has not been identified. This study examines the effect of radiation (10 to 30 Gy) on neutrophil function in a rabbit wound cylinder model. At 3 to 12 weeks after radiation, subcutaneous wound cylinders were implanted in both irradiated and control fields in 19 rabbits. Wound neutrophils were subsequently assayed for phagocytosis ( 3 H-labeled Staphylococcus aureus assay), superoxide production (cytochrome c reduction assay), and surface Mac-1 expression (flow cytometric assay using MHM 23 monoclonal antibody). Phagocytosis of 3 H-labeled S. aureus was significantly lower in neutrophils from irradiated fields compared with controls at 6 and 12 weeks after radiation (6.5 versus 18.9 bacteria per neutrophil at 12 weeks ; p = 0.027). Stimulated neutrophils from irradiated tissue could not increase superoxide production or Mac-1 expression as much as controls, with differences increasing as postirradiation time increased. The diminished phagocytosis, superoxide production, and Mac-1 expression provide a cellular mechanism that may account for susceptibility to infection and poor healing in irradiated tissues.

Background and Purpose. Microamperage direct current and microamperage electromagnetic stimulation are used to accelerate healing in bone. Although many clinicians are using microamperage stimulation to relieve pain and facilitate wound healing, there is significant question regarding whether this low-intensity direct current significantly accelerates soft tissue wound repair. The purpose of this study was to determine whether low-voltage pulsed microamperage current (100 μA, 60 V, 0.1 Hz) enhances the healing of superficial, full-thickness, or incisional wounds created to simulate acute abrasions, ulcers, and lacerations. Subjects. Ten adult Yucatan mini pigs served as the subjects for this study. Methods. Variables associated with healing were measured in 60 matched pairs of surgically induced partial-thickness, full-thickness, and incisional wounds after either sham or anodal (positive-polarity) stimulation with pulsed microamperage current (100 μA) was administered for 1 hour per day for 5 days. Sterile, disposable electrodes (2×4 cm) were placed over each wound, which was kept wrapped and protected throughout the study. Results. At 7 days postinjury, all wounds were healing well with no signs of infection. There were no differences in tensile strength, collagen density, maturity, or deposition (hydroxyproline), wound size, or visual appearance between the sham treatment and treatment lesions. No changes in local subcutaneous oxygen or temperature were found in the swine during or after microamperage stimulation. Conclusion and Discussion. This study did not provide any evidence to support the use of microamperage stimulation to accelerate wound healing. No negative effects, however, were found. Further research is needed to determine whether there is a critical interaction between the size of the electrode relative to the wound, the density of the current, the duration of the treatment, the polarity of the treatment electrode, and the acuity or chronicity of wounding and the effectiveness of microcurrent stimulation for wound healing.

Hypothesis Anti-inflammatory corticosteroids significantly impair wound healing. Retinoids partially, but significantly, reverse this effect. Little is known about the mechanism of steroid retardation or retinoid reversal. We hypothesized that corticosteroids lower transforming growth factor-β (TGF-β) and insulin-like growth factor-I (IGF-I) levels and tissue deposition in wounds and that retinoids stimulate corticosteroid-impaired TGF-β and IGF-I release and collagen production. Design Randomized controlled trial. Setting Wound healing research laboratory. Participants Animal study. Interventions Four wire mesh wound cylinders were implanted subcutaneously into the backs of 72 male Sprague-Dawley rats. Wound healing was impaired by a single subcutaneous injection of 6 mg of methylprednisolone acetate (Depo-Medrol). Two preparations of retinoids were used in separate experiments: all- trans -retinoic acid and 9- cis -retinoic acid that were fed orally. Main Outcome Measures Hydroxyproline content was measured in the healing tissue and TGF-β and IGF-I levels were analyzed in the wound fluid. Results Methylprednisolone treatment significantly decreased TGF-β and IGF-I levels in the wound fluid and hydroxyproline content in the tissue ( P trans - and 9- cis -retinoic acid partially reversed the TGF-β and IGF-I decrease and significantly increased hydroxyproline content toward normal levels ( P trans -retinoic acid enhanced collagen deposition, TGF-β and IGF-I levels over normal chow fed control animals ( P Conclusions Steroids and retinoids have antagonistic effects on growth factors and collagen deposition in wound healing. These effects can be relevant for treatment options in a clinical setting.

Premature infants and neonates are vulnerable to bacterial sepsis. This susceptibility may be due to the relative immaturity of their immune systems. To determine if neonates and, in particular, premature infants have decreased polymorphonuclear leukocyte (PMN) phagocytosis, we tested PMN phagocytosis of Staphylococcus aureus as a function of gestational age in the fetal lamb model. Because phagocytosis is made more efficient by the presence of opsonins in plasma, fetal and postnatal PMN phagocytosis were also measured after exposure to fetal and adult plasma. PMNs were isolated from fetal lambs at 104, 114, 124, and 141 days' gestation (term gestation for the fetal lamb is 145 days), as well as from 10-day-old neonatal sheep and adult sheep. Labeled S aureus were opsonized by incubation in either fetal or adult plasma, or left unopsonized for baseline values. Phagocytosis was measured as a percent of adult PMN phagocytosis after adult plasma opsonization. It was found that fetal PMN function is limited by two factors during the early third trimester: a primary defect in the ability of the PMN to phagocytose S aureaus despite adequate opsonization, and the diminished ability of autologous fetal plasma to opsonize bacteria. The defect in PMN phagocytosis disappears late in the third trimester, but the inability of the fetal plasma to opsonize effectively continues until after birth.

Chemoattractant activity for irradiated and nonirradiated rabbit skin fibroblast and bovine aortic arch endothelial cells was assayed in rabbit wound fluid and sera using a modification of the agarose well method originally described for polymorphonuclear leukocytes. Both serum and wound fluid contained chemoattractants for fibroblasts and endothelial cells. Fibroblast migration was decreased by 70 to 80% when the serum or wound fluid was heated to 56 degrees C for 30 min while endothelial cell migration was reduced by 50 to 60%. Platelet-poor plasma-derived serum had no directive effect on the migration of either cell type.

We compared simultaneous healing processes in fetal, newborn, and maternal rabbits using a miniaturized wound cylinder of expanded Gore-Tex tubing. The tubing was placed subcutaneously in fetal and maternal rabbits on day 23 of pregnancy (term = 31 to 32 days), and in 7-day-old newborn rabbits. At specific intervals, the tubing was removed and analyzed for hydroxyproline accumulation, histology, and cellular enzyme-histochemistry. Granulation tissue ingrowth and accumulation of hydroxyproline were each inversely related to age (fetus greater than newborn greater than maternal). The fetus showed an impressive infiltration of macrophages by day 4, fibroblasts by day 7, and a conspicuous lack of neutrophils in all specimens. Newborns and mothers had few cells until day 7, when a mixture of macrophages, neutrophils, and some fibroblasts appeared. We conclude that the fetus heals wounds rapidly by both mesenchymal cell proliferation and collagen deposition, and that these processes are more rapid in fetuses than in newborn or adult animals despite relative fetal hypoxemia.