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.

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.

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 microM). 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.

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.

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)

Interleukin-1beta 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.