M. Zamirul Hussain

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‐1β, a long‐term elevation of VECF (2×) and transforming growth factor‐β1 (2–3×), 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)

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 pO2 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.

Lactate elicits vascular endothelial growth factor from macrophages: a possible alternative to hypoxia

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.

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.

Removal of intracanal smear by doxycycline in vitro

UNLABELLED Cleansing and shaping result in a smear layer on the instrumented canal wall surfaces. The smear layer may inhibit close contact between sealers and dentin, and inhibits diffusion of medicaments. OBJECTIVE This study assessed the effect of doxycycline hydrochloride (DH) on smear layer on intracanal walls. STUDY DESIGN Scanning electron microscopy was used to evaluate the remaining smear layer using different concentrations of DH. Single-canal palatal roots of extracted maxillary molars were irrigated with saline-15% EDTA; saline-25 mg/ml DH; saline-50 mg/ml DH; saline-100 mg/ml DH; NaOCl-15% EDTA; NaOCl-25 mg/ml DH; NaOCl-50 mg/ml DH; and NaOCl-100 mg/ml DH. The roots were fractured into halves and the amount of smear layer assessed in the middle and apical third. RESULTS Doxycycline-HCl of 100 mg/ml was the most effective in removing smear layer. In the saline group, 100 mg/ml of DH was more effective than EDTA. In the hypochlorite group, 50 mg/ml and 100 mg/ml of DH were more effective than EDTA (p < 0.05). CONCLUSION Doxycycline solution may be an effective irrigant.

Interleukin-1β Activity and Collagen Synthesis in Human Dental Pulp Fibroblasts

Immunopathologic reactions play a significant role in inflammatory diseases of dental pulp. Interleukin-1beta (IL-1beta) is recognized as a key player in mediating cellular immune response. In this study, we measured the content of IL-1beta and its effect on collagen synthesis in cultures of fibroblasts derived from healthy and diseased dental pulps. We found that diseased pulp fibroblasts contain 2.5-fold greater amounts of IL-1beta and synthesized 80% greater amounts of collagen compared with healthy pulp fibroblasts. However, exogenous IL-1beta failed to stimulate collagen synthesis by diseased fibroblasts, whereas collagen synthesis by healthy pulp fibroblasts was stimulated by more than 2-fold. These observations imply that pulp disease induces abnormalities associated with fibroblast response toward IL-1beta.

Involvement of superoxide in the paraquat-induced enhancement of lung collagen synthesis in organ culture

Exposure to paraquat in vivo results in increased synthesis and deposition of collagen. We examined collagen synthesis in organ cultures of neo-natal rat lungs in the presence of paraquat. Paraquat markedly increased the synthesis and accumulation of collagen in these cultures. This effect was abolished by superoxide dismutase. Our studies suggest that the mechanism of paraquat mediated increase in collagen synthesis may involve superoxide.

Role of ADP-ribosylation in wound repair. The contributions of Thomas K. Hunt, MD

Nearly 36 years ago Thomas K. Hunt, with Patrick Twomey, was the first to report that the level of lactate significantly increases in healing wounds. This observation convinced him that lactate, besides being the by‐product of glycolysis, must have a regulatory role in the healing process. He set out to investigate this observation and found it to be so. This article is written in recognition of his foresight. It summarizes the salient findings emanating from this fundamental observation and describes the biochemical principles by which most of the lactate action may be explained. Down‐regulation of the ubiquitous protein modification reaction called ADP‐ribosylation turned out to be a basic signal behind the role of lactate in wound healing. (WOUND REP REG 2003;11:439–444)

Poly(ADP-ribose) synthetase activity during bleomycin-induced lung fibrosis in hamsters

Bleomycin damages cellular DNA and is a potent inducer of pulmonary fibrosis. It has been shown to act through a superoxide-mediated mechanism. We are interested in determining the biochemical mechanisms involved in fibrosis and in this preliminary study we have examined the temporal relationship between early biochemical events associated with DNA damage and fibrosis, in lungs of hamsters after administration of 0.75 unit of bleomycin. The activities of poly(ADP-ribose) synthetase, an enzyme associated with DNA repair, inducible superoxide dismutase (SOD) and prolyl hydroxylase as well as the tissue levels of NAD+ and hydroxyproline in the lung were determined. All three enzyme activities expressed as per milligram DNA or per lung, increased upon bleomycin treatment over the saline-administered controls. Lung poly(ADP-ribose) synthetase activity which is sensitive to DNA breaks, increased first (24% over control in 1 day, P less than 0.0001), attained the maximum value on the 5th day (952% over control, P less than 0.0001), and started to decline thereafter and approached near the control value on 14th day. Bleomycin treatment induced a rapid change in the level of lung NAD+. After 1 day the level of NAD+ was reduced by 42% compared to the control (P less than 0.001), further declined to 65% (P less than 0.001) on the 3rd day, and stayed at that level until the 7th day. On the 14th day, however, the NAD+ level was still lower (29%, P less than 0.05) but approaching the value in the control animals. The activity of prolyl hydroxylase showed significant increase on the 3rd day (50% over control, P less than 0.0001) after bleomycin administration. The enzyme activity continued to increase until the end of the experiment (490% of control, P less than 0.0001, on Day 14). The content of undialyzable hydroxyproline, a marker for collagen, was also increased significantly in the lung tissue on the 3rd day (30% over control, P less than 0.05), continued to increase and reached the highest level on the 14th day (71% over control, P less than 0.001). A significant increase in the activity of SOD (19% over control, P less than 0.001) was seen on the 5th day which continued to increase and attained the highest value on Day 14 (115% over control, P less than 0.0001).(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulation of fibroblast proliferation by lactate‐mediated oxidants

Lactate accumulation is a characteristic of wounds in which glycolysis, occurring both aerobically and anaerobically, contributes to its production. Cell proliferation is a critical component of healing wounds. Recently it has been shown that lactate can chelate iron and thus promotes production of hydroxyl radicals. We report here that exogenous lactate increases intracellular oxidants and that the oxidants promote cell growth in cultured dermal fibroblasts in a dose‐dependent manner. The production of lactate‐mediated oxidant requires iron and hydrogen peroxide and with increasing iron concentration oxidant production is raised as well. However, we found cell proliferation is retarded by 15 mM lactate in the presence of a high iron concentration (7.25 µM). The antioxidants catalase and mannitol abolish the inhibitory effect of high lactate. We conclude from these results that increased proliferation of cultured human fibroblasts by exogenous lactate is mediated by oxidant production.