The role of oxygen during fracture healing.

Oxygen affects the activity of multiple skeletogenic cells and is involved in many processes that are important for fracture healing. However, the role of oxygen in fracture healing has not been fully studied. Here we systematically examine the effects of oxygen tension on fracture healing and test the ability of hyperoxia to rescue healing defects in a mouse model of ischemic fracture healing. Mice with tibia fracture were housed in custom-built gas chambers and groups breathed a constant atmosphere of 13% oxygen (hypoxia), 21% oxygen (normoxia), or 50% oxygen (hyperoxia). The influx of inflammatory cells to the fracture site, stem cell differentiation, tissue vascularization, and fracture healing were analyzed. In addition, the efficacy of hyperoxia (50% oxygen) as a treatment regimen for fracture nonunion was tested. Hypoxic animals had decreased tissue vascularity, decreased bone formation, and delayed callus remodeling. Hyperoxia increased tissue vascularization, altered fracture healing in un-complicated fractures, and improved bone repair in ischemia-induced delayed fracture union. However, neither hypoxia nor hyperoxia significantly altered chondrogenesis or osteogenesis during early stages of fracture healing, and infiltration of macrophages and neutrophils was not affected by environmental oxygen after bone injury. In conclusion, our results indicate that environmental oxygen levels affect tissue vascularization and fracture healing, and that providing oxygen when fractures are accompanied by ischemia may be beneficial.

Optimization of clinical breast examination.

BACKGROUND: Breast examination is necessary for evaluation of the 8% to 17% of cancers missed by mammograms, but it is being done less often and less effectively. To improve the use of breast examination, we tested whether a technique to focus attention could improve the call rate (the percent of examinations leading to further evaluation), a measure of quality, without retraining in examination technique. METHODS: Clinicians were randomized to complete 1 of 2 dedicated, de-identified forms after routine breast examination: a long form intended to focus attention by requesting general breast descriptors along with clinical information and breast examination findings (10 clinicians recorded 964 examinations) or a short form recording only clinical information and examination findings (11 clinicians recorded 558 examinations). There was no technique retraining. Study call rates were compared with historical controls (298 breast examinations by 16 clinicians). RESULTS: The call rates by the study groups of clinicians were similar, but the call rate using either form (8.3%) was significantly higher than the call rate in the preceding year when no dedicated form was used (4.7%; P=.031), suggesting a Hawthorne effect in which altering conditions of data collection (using the dedicated forms) functioned as an independent variable. Surveillance, Epidemiology, and End Results data predicted 3.4 cancers in all 1822 patients; 4 cancers were found. CONCLUSION: Breast examination call rate doubled when attention was focused on examination results using a dedicated form, and we found the anticipated cancers. Breast examination quality can be improved by focusing clinician attention without retraining in technique.

Hyperbaric oxygen stimulates vasculogenic stem cell growth and differentiation in vivo.

We hypothesized that oxidative stress from hyperbaric oxygen (HBO(2), 2.8 ATA for 90 min daily) exerts a trophic effect on vasculogenic stem cells. In a mouse model, circulating stem/progenitor cell (SPC) recruitment and differentiation in subcutaneous Matrigel were stimulated by HBO(2) and by a physiological oxidative stressor, lactate. In combination, HBO(2) and lactate had additive effects. Vascular channels lined by CD34(+) SPCs were identified. HBO(2) and lactate accelerated channel development, cell differentiation based on surface marker expression, and cell cycle entry. CD34(+) SPCs exhibited increases in thioredoxin-1 (Trx1), Trx reductase, hypoxia-inducible factors (HIF)-1, -2, and -3, phosphorylated mitogen-activated protein kinases, vascular endothelial growth factor, and stromal cell-derived factor-1. Cell recruitment to Matrigel and protein synthesis responses were abrogated by N-acetyl cysteine, dithioerythritol, oxamate, apocynin, U-0126, neutralizing anti-vascular endothelial growth factor, or anti-stromal cell-derived factor-1 antibodies, and small inhibitory RNA to Trx reductase, lactate dehydrogenase, gp91(phox), HIF-1 or -2, and in mice conditionally null for HIF-1 in myeloid cells. By causing an oxidative stress, HBO(2) activates a physiological redox-active autocrine loop in SPCs that stimulates vasculogenesis. Thioredoxin system activation leads to elevations in HIF-1 and -2, followed by synthesis of HIF-dependent growth factors. HIF-3 has a negative impact on SPCs.

Lactate stimulates vasculogenic stem cells via the thioredoxin system and engages an autocrine activation loop involving hypoxia-inducible factor 1.

The recruitment and differentiation of circulating stem/progenitor cells (SPCs) in subcutaneous Matrigel in mice was assessed. There were over one million CD34(+) SPCs per Matrigel plug 18 h after Matrigel implantation, and including a polymer to elevate the lactate concentration increased the number of SPCs by 3.6-fold. Intricate CD34(+) cell-lined channels were linked to the systemic circulation, and lactate accelerated cell differentiation as evaluated based on surface marker expression and cell cycle entry. CD34(+) SPCs from lactate-supplemented Matrigel exhibited significantly higher concentrations of thioredoxin 1 (Trx1) and hypoxia-inducible factor 1 (HIF-1) than cells from unsupplemented Matrigel, whereas Trx1 and HIF-1 in CD45(+) leukocytes were not elevated by lactate. Results obtained using small inhibitory RNA (siRNA) specific to HIF-1 and mice with conditionally HIF-1 null myeloid cells indicated that SPC recruitment and lactate-mediated effects were dependent on HIF-1. Cells from lactate-supplemented Matrigel had higher concentrations of phosphorylated extracellular signal-regulated kinases 1 and 2, Trx1, Trx reductase (TrxR), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1 (SDF-1) than cells from unsupplemented Matrigel. SPC recruitment and protein changes were inhibited by siRNA specific to lactate dehydrogenase, TrxR, or HIF-1 and by oxamate, apocynin, U0126, N-acetylcysteine, dithioerythritol, and antibodies to VEGF or SDF-1. Oxidative stress from lactate metabolism by SPCs accelerated further SPC recruitment and differentiation through Trx1-mediated elevations in HIF-1 levels and the subsequent synthesis of HIF-1-dependent growth factors.

Lactate modulates gene expression in human mesenchymal stem cells.

PURPOSE: Surgical wounds are characterised by elevated tissue lactate concentrations. This accumulated lactate is capable of stimulating collagen synthesis and new vessel growth as well. Recently, it has been shown in vivo that lactate is also able to favour homing of stem cells. The aim of this investigation was to test the hypothesis that lactate has an impact on gene expression of mesenchymal stem cells (MSC). MATERIALS AND METHODS: MSC were isolated from human bone marrow using the density gradient technique and incubated with alpha-methoxyethoxymethyl containing 10% fetal calf serum at 37 degrees C under 95% air and 5% CO(2). Cultured MSC were characterised by in vitro differentiation assays and fluorescence-activated cell sorting (FACS) analysis. Characterised MSC were treated with 15 mM lactate for different time periods (1, 6 and 24 h and 3 and 7 days). Gene expression analysis was performed using a custom-designed oligonucleotide microarray. A significant alteration of gene expression was defined as a two-fold stimulation or inhibition. The phenotype of MSC was investigated by FACS analysis of specific surface epitope patterns. RESULTS: Gene expression analysis shows 63 up- and 51 down-regulated genes after 1 h of treatment, 45 up- and 47 down-regulated genes after 6 h of treatment, 57 up- and 72 down-regulated genes after 24 h of treatment, 103 up- and 28 down-regulated genes after 3 days of treatment and 50 up- and 101 down-regulated genes after 7 days of treatment with lactate. The majority of the modulated genes are related to the expression of cytokines, transcription factors and cell-cycle- or cellular-matrix-associated proteins. In particular, lactate up-regulates the expression of interleukin-6 (3 days, 4.11-fold), of heat shock protein 70 (3 days, 2.36-fold) and of hypoxia-inducible factor-1alpha (3 days, 2.09-fold). A down-regulating effect of lactate is observed for superoxide dismutase 2 (1 h, 0.5-fold; 24 h, 0.4-fold; 7 days, 0.32-fold) and BCL2-associated X protein (24 h, 0.42-fold; 7 days, 0.4-fold). Expression of cell surface antigens clusters of differentiation 29, 44, 59, 73, 90, 105, 106 and 146 does not change over the time period of lactate treatment. CONCLUSIONS: Lactate modulates expression of genes involved in wound healing. However, lactate does not profoundly change the phenotype of MSC. In addition to providing new insights into the wound healing physiology, these data could also be the rationale for new treatment strategies for chronic non-healing wounds.

Lactate, with oxygen, incites angiogenesis.

Lactate has been reconsidered! As we now know, most is produced aerobically We report that lactate accumulation commonly occurs in the presence of oxygen and is sufficient to instigate signals for angiogenesis and connective tissue deposition. These include vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF beta), interleukin-1 (IL-1), and hypoxia-inducible factor (hif-1alpha). This paper, a mini-review, is occasioned by new data showing increased presence of VEGF and angiogenesis in an oxygenated site by adding a slow-release source of lactate into Matrigel and implanting the Matrigel subcutaneously in mice.

Aerobically derived lactate stimulates revascularization and tissue repair via redox mechanisms.

Hypoxia serves as a physiologic cue to drive an angiogenic response via HIF-dependent mechanisms. Interestingly, minor elevation of lactate levels in the tissue produces the same effect under aerobic conditions. Aerobic glycolysis contributes to lactate accumulation in the presence of oxygen, especially under inflammatory conditions. We previously postulated that aerobic lactate accumulation, already known to stimulate collagen deposition, will also stimulate angiogenesis. If substantiated, this concept would advance understanding of wound healing and aerobic angiogenesis because lactate accumulation has many aerobic sources. In this study, Matrigel plugs containing a powdered, hydrolyzable lactate polymer were implanted into the subcutaneous space of mice. Lactate monomer concentrations in the implant were consistent with wound levels for more than 11 days. They induced little inflammation but considerable VEGF production and were highly angiogenic, as opposed to controls. Arterial hypoxia abrogated angiogenesis. Furthermore, inhibition of lactate dehydrogenase by using oxamate also prevented the angiogenic effects of lactate. Lactate monomer, at concentrations found in cutaneous wounds, stabilized HIF-1alpha and increased VEGF levels in aerobically cultured human endothelial cells. Accumulated lactate, therefore, appears to convey the impression of "metabolic need" for vascularization, even in well-oxygenated and pH-neutral conditions. Lactate and oxygen together stimulate angiogenesis and matrix deposition.

Lactate stimulates endothelial cell migration.

The significance of the high lactate levels that characterize healing wounds is not fully understood. Lactate has been shown to enhance collagen synthesis by fibroblasts and vascular endothelial growth factor (VEGF) production by macrophages and endothelial cells. VEGF has been shown to induce endothelial cell migration. However, it has not been shown whether accumulated lactate correlates with the biological activity of VEGF. Therefore, we investigated the effect of lactate on migration of endothelial cells. Human umbilical vein endothelial cells and human microvascular endothelial cells were cultured to subconfluent monolayers in standard six-well tissue culture plates. Following a 24-hour serum starvation, cells were treated with the indicated concentrations of l-lactate. Cell migration was assessed using a modified Boyden chamber. VEGF protein in the cell culture supernatant was measured by enzyme-linked immunoassay. Lactate-enhanced VEGF protein synthesis in a time- and dose-dependent manner. Lactate added into the bottom well did not stimulate cellular migration from the upper well. However, lactate when added together with endothelial cells to the bottom well of the Boyden chamber increased cellular migration in a dose-dependent manner. This effect was blocked by anti-VEGF and by cycloheximide. Lactate enhances VEGF production in endothelial cells, although lactate, itself, is not a chemoattractant. We conclude that the lactate-mediated increase in cellular migration is regulated by VEGF.

Using physiology to improve surgical wound outcomes.

Despite major advances in surgical management and approaches, including aseptic techniques, prophylactic antibiotics, and laparoscopic surgery, surgical wound infection and wound failure remain common complications of surgery. In a review of the literature, the authors found that a growing body of literature supports the concept that patient factors are a major determinant of wound outcome after surgery. In particular, wounds are exquisitely sensitive to hypoxia, which is both common and preventable. Perioperative management can be adapted to promote postoperative wound healing and resistance to infection. The most important factors are fluid management, temperature management, pain control, increased arterial oxygen tension, and, as has been long recognized, appropriate sterile techniques and administration of prophylactic antibiotics. This article reviews how knowledge of and attention to physiology can improve quality of care in both acute and chronic wounds.

IGF-I-induced VEGF expression in HUVEC involves phosphorylation and inhibition of poly(ADP-ribose)polymerase.

Insulin-like growth factor-I (IGF-I) has been shown to promote angiogenesis by enhancing vascular endothelial growth factor (VEGF) expression. However, how IGF-I-induces VEGF expression is not yet fully understood. With this investigation, we propose a new possible mechanism involving downregulation of poly(ADP-ribosyl)ation (pADPR). We first demonstrated that IGF-I increased VEGF protein expression in endothelial cells. Inhibitors of mitogen activated kinase (PD 98059), phosphatidyl-3-inositol-kinase (LY 294002), and protein kinase C (staurosporine) diminished the IGF-I effect suggesting the involvement of signal transduction. Since there is an established link between pADPR and transcriptional activity, we focused on a possible role of poly(ADP-ribose)polymerase (PARP). The inhibition of PARP by 3-aminobenzamide or nicotinamide enhanced VEGF expression. Additionally, IGF-I markedly decreased PARP activity. Furthermore, the IGF-I-mediated inhibition of PARP could be demonstrated as a result of protein phosphorylation since phosphorylation of PARP decreased its activity in vitro and IGF-I treatment of endothelial cells induced PARP phosphorylation. The IGF-I-mediated phosphorylation and inhibition of PARP represent a novel mechanism of VEGF protein expression.

Dermal wound healing is subject to redox control.

Previously we have reported in vitro evidence suggesting that that H2O2 may support wound healing by inducing VEGF expression in human keratinocytes (C. K. Sen et al., 2002, J. Biol. Chem.277, 33284-33290). Here, we test the significance of H2O2 in regulating wound healing in vivo. Using the Hunt-Schilling cylinder approach we present the first evidence that the wound site contains micromolar concentrations of H2O2. At the wound site, low concentrations of H2O2 supported the healing process, especially in p47(phox)- and MCP-1-deficient mice in which endogenous H2O2 generation is impaired. Higher doses of H2O2 adversely influenced healing. At low concentrations, H2O2 facilitated wound angiogenesis in vivo. H2O2 induced FAK phosphorylation both in wound-edge tissue in vivo and in human dermal microvascular endothelial cells. H2O2 induced site-specific (Tyr-925 and Tyr-861) phosphorylation of FAK. Other sites, including the Tyr-397 autophosphorylation site, were insensitive to H2O2. Adenoviral gene delivery of catalase impaired wound angiogenesis and closure. Catalase overexpression slowed tissue remodeling as evidenced by a more incomplete narrowing of the hyperproliferative epithelium region and incomplete eschar formation. Taken together, this work presents the first in vivo evidence indicating that strategies to influence the redox environment of the wound site may have a bearing on healing outcomes.

Hyperoxia and angiogenesis.

We hypothesized that tissue hyperoxia would enhance and hypoxia inhibit neovascularization in a wound model. Therefore, we used female Swiss-Webster mice to examine the influence of differential oxygen treatment on angiogenesis. One milliliter plugs of Matrigel, a mixture of matrix proteins that supports but does not itself elicit angiogenesis, were injected subcutaneously into the mice. Matrigel was used without additive or with added vascular endothelial growth factor (VEGF) or anti-VEGF antibody. Animals were maintained in hypoxic, normoxic, or one of four hyperoxic environments: hypoxia -- 13 percent oxygen at 1 atmosphere absolute (ATA); normoxia -- 21 percent oxygen at 1 ATA; hyperoxia -- (groups a-d) 100 percent oxygen for 90 minutes twice daily at the following pressures: Group a, 1 ATA; Group b, 2 ATA; Group c, 2.5 ATA; Group d, 3.0 ATA. Subcutaneous oxygen tension was measured in all groups. The Matrigel was removed 7 days after implantation. Sections were graded microscopically for the extent of neovascularization. Angiogenesis was significantly greater in all hyperoxic groups and significantly less in the hypoxic group compared with room air-exposed controls. Anti-VEGF antibody abrogated the angiogenic effect of both VEGF and increased oxygen tension. We conclude that angiogenesis is proportional to ambient pO(2) over a wide range. This confirms the clinical impression that angiogenesis requires oxygen. Intermittent oxygen exposure can satisfy the need for oxygen in ischemic tissue.

Oxygen: from the benefits of inducing VEGF expression to managing the risk of hyperbaric stress.

Hypoxia limits wound healing. Both normobaric (1 atm) and hyperbaric oxygen (HBO) approaches have been used clinically to oxygenate wound tissue. Recently, we reported that HBO ameliorates stress-induced impairment of dermal healing. We examined the effect of pressure on oxygen-induced vascular endothelial growth factor (VEGF) expression by human HaCaT keratinocytes. Next, we investigated the effect of HBO on whole-body redox and on the ratio of oxidized to reduced glutathione (GSSG/GSH) in the liver, heart, lung, and brain of rats. Superoxygenation (90% O2) of keratinocytes partially arrested cell growth. G2-M growth arrest was substantially augmented by HBO. HBO also caused apoptosis in a small subpopulation. Normobaric oxygen, but not HBO (2 atm), potently induced the expression of VEGF165 and 189. In vivo electron paramagnetic resonance spectroscopy imaging revealed a clear shift of the whole-body redox status toward oxidation in response to HBO. The standard diet of laboratory rats contains excessive (17x human recommended dietary allowance) alpha-tocopherol (E++), which confers exceptional resistance to oxidant insults. People with chronic wounds commonly suffer from under- or malnutrition. We generated vitamin E-deficient (E-) rats by long-term dietary vitamin E restriction. HBO did not raise GSSG/GSH in E++ rats, but post-HBO GSSG/GSH was significantly higher in E- compared with E++. Thus, rats on antioxidant-enriched diet were well protected against HBO. The risk of oxidative stress may negatively impact the net benefits of HBO. This is of special concern for people with inadequate intake of dietary antioxidants. Nutritional antioxidant supplementation may offset HBO-induced oxidative stress.

Oxygen: at the foundation of wound healing--introduction.

"Wound Healing: Oxygen & Emerging Therapeutics" Columbus, Ohio, September 12-15, 2002. Sponsored by the National Institutes of Health (R13 AR 049171), International Union of Biochemistry & Molecular Biology and UNESCO-Global Network of Molecular & Cell Biology. Conference co-chairs: Chandan K. Sen, the Ohio State University Medical Center and Thomas K. Hunt, University of California-San Francisco. This congress was conceived for two reasons: to consolidate what is known about oxygen in the repair process and to stimulate discussion about new developments of control of healing by redox regulated signaling processes. A historical and evolutionary perspective on the role of oxygen in wound healing--from the classical physiology of oxygen in the wound to the refined concept of redox signaling--is presented.

Lactate and oxygen constitute a fundamental regulatory mechanism in wound healing.

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)

Age-dependency of insulin-like growth factors, insulin-like growth factor-binding proteins, and acid labile subunit in plasma and wounds of surgical patients.

Wound problems are common in the elderly. We hypothesized that age-related decrements in blood levels of components of the insulin-like growth factor (IGF) system are reflected in the wound environment. In this prospective, observational study IGF-I, IGF-II, IGF-binding protein-2, IGF-binding protein-3, and acid labile subunit were measured by immunoassays in the wound fluid and plasma of young (23.5 +/- 3.3 years) and elderly (78.9 +/- 6.2 years) patients before and daily for 4 days after elective surgery. IGFs, IGFBP-3, and acid labile subunit in plasma were significantly lower in the elderly group (p < 0.0001). The decrements of these proteins in plasma were reflected in corresponding decrements of 25-70% in the wound fluid of elderly patients (p < 0.0001). Additionally, bioavailability of IGF-I was less in the aged. The IGF parameters in the wound displayed a constant ratio with those of blood, suggesting that blood contributes a major share of the IGF that enters the wound during the initial phase of healing. The current data adds to accumulating evidence that a decline in the IGF system in aged patients contributes to the healing deficits observed in the elderly.

Correlation of a simple direct measurement of muscle pO(2) to a clinical ischemia index and histology in a rat model of chronic severe hindlimb ischemia.

PURPOSE: The lack of suitable experimental models of chronic severe limb ischemia and deficiencies in the available methods that allow for direct intermittent measurement of regional limb perfusion are obstacles to the evaluation of recently developed molecular strategies to reverse severe limb ischemia. Our aim was to develop a model of clinically relevant severe limb ischemia and correlate a simple direct measurement of muscle pO(2) to a clinical ischemia index, muscle mass, and capillary density. METHODS: Severe hindlimb ischemia was induced in 44 adult rats with ligation of the left common iliac artery, the femoral artery, and their branches. The effect of ischemia on muscle pO(2) was measured in the left gastrocnemius with room air and with 100% oxygen at 3, 10, 24, and 40 days after ischemia was induced. Clinical ischemia index, muscle mass, cellular proliferation, and capillary density also were assessed. RESULTS: The clinical ischemia index of the left limb was most severe at day 10, with evidence of pressure sores, a pale and dusky limb, and abnormal gait. With the rats breathing room air, muscle pO(2) was significantly lower in the left limbs than in the right limbs at days 3, 10, 24, and 40. After an oxygen challenge (100% O(2)), muscle pO(2) was significantly lower at 3, 10, and 40 days. At 3 days, the fraction of muscle mass per total body weight of the left tibialis anterior (TA) was significantly greater than the right TA as a result of edema and inflammation. By days 10, 24, and 40, the left gastrocnemius and TA masses were significantly less than the right as a result of muscle atrophy. Histopathology showed severe necrosis in the left gastrocnemius and TA on day 3. Inflammation was greatest by day 10. Necrotic muscle regenerated but remained atrophic at 40 days. The TA was slower to recover than the gastrocnemius. Capillary densities and capillary-to-muscle fiber ratios were greater in the ischemic limb than in the normal limb at day 24. Cellular proliferation as determined with bromodeoxyuridine labeling reagent staining was maximal in the ischemic limb at day 3. CONCLUSION: We have developed a rat model of chronic severe hindlimb ischemia with persistent ischemia as shown with a simple direct measurement of muscle pO(2) for up to 40 days. This model of severe hindlimb ischemia may be applicable for future studies of molecular strategies to treat severe limb ischemia in humans.