Lactate induces collagen synthesis, VEGF expression and migration in endothelial cells via generation of superoxide

Abstract

Introduction: Surgical wounds are characterized by increased tissue lactate concentrations. As we know now, accumulated lactate in wounds is not a by-product of hypoxia or anaerobic glycolysis in wound healing. In fact, lactate is mostly produced aerobically. Lactate accumulation commonly occurs in the presence of oxygen, especially under inflammatory conditions, and is capable of initiating signals for angiogenesis and connective tissue deposition. The significance of the high lactate levels that characterize healing wounds is not fully understood yet. Therefore, the aim of this study was to investigate the effect of lactate on migration and collagen synthesis in endothelial cells. Material and Methods: Human umbilical vein endothelial cells (HUVEC) were incubated with different concentrations of lactate. Gene expression analysis was performed using oligonucleotid-microarrays. Differentially expressed and selected genes of interest were additionally evaluated using qRT-PCR. HPLC was carried out to detect the hydroxyproline-concentration in cell supernatants. The generation of superoxide (O2-) was evaluated in vivo using a nitroblue tetrazolium test (NBT). Cell migration was assessed using a modified Boyden chamber. VEGF expression in cell supernatant was detected via ELISA. Results are given as mean ±SD or as fold-change to the corresponding control. Results: Lactate induced gene expression of collagen-I al (16 and 24 h: 2-fold) and collagen-IV al mRNA (4 h: 2.5-fold). Similarly, we detected high concentrations of hydroxyproline in the supernatants of lactate stimulated cells (72 h: control 0.20+0.04 vs. lactate 10 mM 0.32+0.02^* vs. Laktat 15 mM 0.32+0.03^* μg/ml hydroxyproline; ^*p<0.05). Endothelial migration was increased by the supernatant of lactate stimulated HUVEC in a time-dependent manner (4 h: 37+17%^* and 8 h: 52+16%^* vs. control; ^*p<0.05). Concerning lactate stimulated HUVEC, microarray analysis revealed enhanced expression of NADPH oxidaes (NOX-4) (1 h: 7-fold; 8 h: 4-fold), a diminished expression of thioredoxin (Trx) (1 h: 4-fold; 4 h: 3-fold) and superoxide dismutase 2 (SOD2) (4 h: 2-fold; 24 h: 8-fold). The NBT in vivo revealed increased formation of formazaan crystals in lactate stimulated HUVEC. A significant overexpression of VEGF in the supernatant was detected supplying HUVEC with an exogenous superoxide-donor (SIN-1, 1mM) (64+21%; p<0.05). This effect was reversed using a NADPH-inhibitor. Conclusion: Lactate enhances collagen production and migration in endothelial cells. We suggest that this detected effect is- at least partially- mediated by increased generation of superoxide radicals via NADPH activity. The correlation between lactate and modulation of the activity of NADPH provides new insights into the wound healing physiology. Hence, this data might serve for new treatment strategies of chronic non-healing wounds.