The excessive growth of a tumor requires high rates of glucose uptake and glycolysis and continuous recruitment of new blood vessels. Here, we provide several lines of evidence showing that pyruvic acid, the end product of glycolysis, exhibits strong angiogenic activity. Pyruvic acid promoted angiogenesis in chorioallantoic membrane assay and in vivo mouse Matrigel plug assay. Pyruvic acid also positively affects angiogenic cascade, DNA synthesis, migration, and tube formation in bovine aortic endothelial cells. Furthermore, mRNA expression of fibroblast growth factor receptor-2 and vascular endothelial growth factor was enhanced by pyruvic acid. These results strongly suggest that pyruvic acid plays an important role in angiogenesis for tumor growth and metastasis.
Angiogenesis, the formation of new blood vessels emerging from preexisting endothelial vasculature (1), plays a crucial role in a wide range of physiological events including embryonic development, placental implantation, and wound healing for the delivery of oxygen and nutrients as well as removal of waste products (2, 3). It is also initiated in response to certain pathological conditions, such as solid tumor growth, diabetic retinopathy, psoriasis, and rheumatoid arthritis in which angiogenesis is responsible for the progression of such diseases (4). Complex and diverse cellular actions are implicated in angiogenesis, such as extracellular matrix degradation, proliferation and migration of endothelial cells, and morphological differentiation of endothelial cells to form tubes (5). Although all of these cascades are strictly regulated under normal conditions, abnormal high vascularization is clearly implicated in tumor growth and metastasis. The extreme growth of tumor larger than a few cubic millimeters in size requires continuous recruitment of new blood vessels (6). These newly synthesized blood vessels also provide a route for cancer cells to enter the blood circulation and spread to other distant organs (7). In addition, one of the most important attributes of the tumor growth is its responsiveness to local metabolic changes, high rates of glucose uptake and glycolysis by up-regulation of pyruvic acid transporter, monocarboxylate transporter, and the alterations in the glycolytic enzyme complex (8). The major end product of this abnormal metabolic pathway, pyruvic acid, is, therefore, produced in quantity and must be eliminated from the cells to permit persistent high glycolytic flux (9). That is, high rates of pyruvic acid production occur in tumor, and this condition may trigger neovascularization. In this report, we investigated whether pyruvic acid modulates angiogenic action of endothelial cells for growth and metastasis of tumor cells. We demonstrated that pyruvic acid markedly promotes new blood vessel formation in CAM3 assay and in vivo Matrigel assay. We also showed that pyruvic acid enhances proliferation, migration, and tube formation. Furthermore, pyruvic acid up-regulated the expression of FGFR-2 and VEGF. Taken together, these results suggest that pyruvic acid facilitate the tumor formation via promoting angiogenesis.
Materials and Methods
Fertilized chick eggs were incubated under conditions of constant humidified egg breeder at 37°C. On the third day of incubation, about 2 ml of egg albumin were aspirated by an 18-gauge hypodermic needle, to detach the developing CAM from the shell. After more incubation for 6 days, sample-loaded thermanox coverslips (Nunc, Naperville, IL) were air-dried and applied to the CAM surface for testing of angiogenesis activation by pyruvic acid. Three days later, 1–2 ml of 10% fat emulsion (Intralipose) was injected into the chorioallantois and observed under a microscope.
Seven-week-old, specific pathogen-free male C57BL/6 mice were supplied from Hyochang Science (Taegu, South Korea). They were provided with autoclaved tap water and lab chow ad libitum and were housed at 23 ± 0.5°C, 10% humidity, in a 12-h light-dark cycle.
In Vivo Matrigel Plug Assay.
C57BL/6 mice (7 weeks of age) were given s.c. injections of 500 μl of Matrigel (Collaborative Biomedical Products, Bedford, MA) at 4°C containing pyruvic acid (Sigma, St. Louis, MO). After injection, the Matrigel rapidly formed a plug. After 7 days, the skin of the mouse was easily pulled back to expose the Matrigel plug, which remained intact. After quantitative differences were noted and photographed, hemoglobin was measured using the Drabkin method and Drabkin reagent kit 525 (Sigma) for the quantitation of blood vessel formation (10). The concentration of hemoglobin was calculated from a known amount of hemoglobin assayed in parallel.
BAECs were grown in DMEM supplemented with heat-inactivated 10% fetal bovine serum (Life Technologies, Grand Island, NY), 100 units/ml penicillin, and 100 μg/ml streptomycin in a 37°C incubator with a humidified atmosphere containing 5% CO2.
[3H]Thymidine Incorporation Assay.
BAECs, grown to near confluence in 24-well culture plates, were made quiescent and treated with pyruvic acid for 48 h. Cells were labeled with [3H]methylthymidine (25mCi/mmol; Amersham, Aylesbury, United Kingdom) for 4 h before the assay. After labeling, unincorporated [3H]methylthymidine was removed by washing with 10% trichloroacetic acid, and then incorporated [3H]methylthymidine was extracted in 0.2 m NaOH and 0.1% SDS at 37°C for 1 h. The cpm values from cultures were counted with a liquid scintillation counter (Beckman Instruments, Fullerton, CA).
Wounding Migration Assay.
BAECs, plated on 60-mm culture dishes at 90% confluence, were wounded with a razor blade 2 mm in width and marked at the injury line. After wounding, the cultures were washed with serum-free medium and further incubated in DMEM with 1% serum, 1 mm thymidine, and/or pyruvic acid. BAECs were allowed to migrate for 4 h and were rinsed with serum-free medium, followed by fixing with absolute methanol and staining with Giemsa. Migration was quantitated with counting the number of cells that moved beyond the reference line.
Tube Formation Assay.
BAECs (5 × 105 cells) were seeded on a layer of previously polymerized Matrigel with or without pyruvic acid. Matrigel culture were incubated at 37°C. After 6 h, changes of cell morphology were captured through a phase-contrast microscope and photographed.
Total RNA from BAECs was isolated using Trizol reagent (Life Technologies) according to the manufacturer’s instructions. First-stranded cDNA was synthesized by Moloney murine leukemia virus reverse transcriptase with 5 μg of each DNA-free total RNA sample and oligo(dT)15 (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. Equal amounts of cDNA were subsequently amplified by PCR in a 50-μl reaction volume containing 1× PCR buffer, 200 μm dNTPs, 10 μm each specific primer, and 1.25 units Taq DNA polymerase (Perkin-Elmer). Amplification products were electrophoresed on 1.5% agarose gels and visualized by ethidium bromide staining under UV transillumination.
Data Analysis and Statistics.
Data are presented as means ± SE or as percentage of control. Statistical comparisons between groups were performed using the Student’s t test. P < 0.05 was considered statistically significant.
Pyruvic Acid Stimulates Angiogenesis in Vivo.
To determine whether pyruvic acid has angiogenic activity, CAM assay was first carried out. After 12 days of incubation, pyruvic acid strongly elicited on angiogenic response, which is visible with the microscope as a spoke-wheel-like pattern of blood vessels. However, around the control thermanox coverslip containing vehicle alone, no growth of new blood vessels was observed (Fig. 1,A). As shown in Fig. 1,D, application of 300 μg of pyruvic acid exhibits 80.7% of positive response. To test whether pyruvic acid induces angiogenesis in a dose-dependent manner, we performed CAM assay with various concentrations (1 to 300 μg/egg) of pyruvic acid. The effect of pyruvic acid on chick embryonic angiogenesis was increased dose dependently. We then evaluated the effect of pyruvic acid on the ongoing angiogenesis process in the in vivo mouse Matrigel plug assay (Ref. 11; Fig. 2). Matrigel and heparin (9 units/500 μl) with or without pyruvic acid (0.3 mg and 0.5 mg, respectively) were s.c. injected into C57BL/6 mice, and 7 days later, the formed Matrigel plug in mice was excised and histologically examined. Plugs with Matrigel alone were pale in their color indicating no or less blood vessel formation. In contrast, plugs mixed with pyruvic acid appeared dark-red color (data not shown). Because the Matrigel treated with 0.5 mg of pyruvic acid contains too many large vessels to perform histological examination, we sectioned only 0.3 mg pyruvic acid treated-Matrigel and control Matrigel plugs. The stained section showed that Matrigel containing pyruvic acid had produced more vessels in gels than Matrigel alone (Fig. 2, A–C). The new vessels were abundantly filled with intact RBCs, which indicates the formation of a functional vasculature inside the Matrigel and blood circulation in newly formed vessels by angiogenesis induced by pyruvic acid. We also measured the hemoglobin content inside the Matrigel plugs to quantify the angiogenesis induced by pyruvic acid. Whereas hemoglobin in control was nearly 0.2 g/dl, pyruvic acid markedly enhanced the hemoglobin quantity to about 19 g/dl (Fig. 2 D). Taken together, we demonstrated that pyruvic acid exhibited a profound angiogenic activity in vivo.
Pyruvic Acid Stimulates Proliferation, Migration, and Tube Formation of BAECs.
These angiogenic activities of pyruvic acid in vivo urged us to confirm the angiogenic effect of pyruvic acid on each step in angiogenesis. We tested the effect of pyruvic acid on angiogenesis in each step by using in vitro angiogenesis assays. We first examined the effect on the BAEC viability by increasing the concentration of pyruvic acid (from 1 to 10 mm). As expected, pyruvic acid did not show any cytotoxic effect on the BAECs (data not shown). In active vascular remodeling phase such as in tumors, endothelial cells proliferate 20 to 2000 times faster than normal endothelium in the adult to form new blood vessels (12). Therefore, we examined the effect of pyruvic acid on DNA synthesis of BAECs by [3H]thymidine incorporation assay. As shown in Fig. 3,A, pyruvic acid significantly increased the DNA synthesis activity of BAECs in a dose-dependent manner. The migration of endothelial cells is one of the critical features in the formation of new blood vessels and in the repair of injured vessels. Therefore, we examined the effect of pyruvic acid on the movement of BAECs from a wound edge in vitro. The wound was made by removing a patch of cells with a razor blade, forming a confluent monolayer. To minimize the possible effects of serum factors, all of the experiments were conducted in DMEM with 1% fetal bovine serum. Pyruvic acid profoundly stimulated the migration of BAECs from the edge of the wound into the open area in a dose-dependent manner. The migratory activity with 10 mm pyruvic acid was about four times higher than that of control. Furthermore, this effect of pyruvic acid is a little higher than that of bFGF (25ng/ml), which is well known to increase the activity of endothelial cell migration (Fig. 3,B). To determine the effect of pyruvic acid on BAEC differentiation, we conducted a tube formation assay (13). BAECs were placed on a growth factor-reduced Matrigel-coated plate and were incubated for 6 h. As shown in Fig. 3 C, whereas BAECs on Matrigel failed to form blood vessel network in the absence of pyruvic acid, pyruvic acid stimulated the formation of many strong tube-like structures. These results further emphasized that pyruvic acid has profound angiogenic actions by stimulating all three of the major angiogenic steps.
Pyruvic Acid Up-Regulated FGFR2 and VEGF mRNA Expression.
To determine what molecules were involved in the angiogenic activity of pyruvic acid, we examined the expression of angiogenic factors and their receptors in BAECs treated with pyruvic acid using RT-PCR. Although bFGF mRNA expression was unchanged, FGFR-2, the receptor of bFGF, was increased by the treatment of pyruvic acid. In addition, the expression of VEGF, one of the strong angiogenic factors, was also markedly up-regulated by treatment with pyruvic acid in a dose-dependent manner (Fig. 4). These data suggest that the up-regulated expression of FGFR2 and VEGF may be responsible for the angiogenic action of BAECs treated with pyruvic acid.
Pyruvic acid is a nontoxic low Mr metabolite that is used worldwide as a drug in commercial diet food. It has lots of benefits such as increasing exercise endurance, promotion of fat loss, and protection of DNA. Furthermore, in the previous molecular studies, tumor angiogenesis and abnormal glycolysis were caused by the multiple genetic alterations that induce tumor growth (14). Independent of this effect, in the present study, we clearly elucidated the angiogenic activity of pyruvic acid which is the end product of abnormal glycolytic metabolism in tumors, by performing in vivo and in vitro angiogenesis assays. We first tested the effect of pyruvic acid on angiogenesis in chick embryo and then observed that pyruvic acid significantly promotes the development of capillary networks in CAM (Fig. 1). Because pyruvic acid is naturally produced acid and high amount of pyruvic acid can cause acidosis in cells, this strong angiogenic effect of pyruvic acid in chick embryo development could have occurred by the action of acidotic pH of pyruvic acid in chick CAM. However, recent study has shown that acidosis inhibits angiogenic behavior, such as migration, proliferation, and differentiation of endothelial cells despite enhanced VEGF and bFGF mRNA expression in BAECs (15). On the basis of these facts, we suggest that angiogenic activity of pyruvic acid in CAM assay is triggered not by acidosis but by its own action as a natural small molecule. We also confirmed the angiogenic activity of pyruvic acid by performing in vivo mouse Matrigel plug assays. Pyruvic acid highly elevated the formation of neovessels in Matrigel, and we noted that these newly synthesized vessels participated actively in the circulating of blood cells in mice (Fig. 2).
The angiogenic process is a tightly regulated phenomenon that includes at least four sequential steps: (a) proliferation of endothelial cells; (b) enzymatic degradation of basement membrane and interstitial matrices by endothelial cells (16, 17); (c) migration of endothelial cells; and (d) formation of capillary loops by endothelial cells (18). We next observed the effects of pyruvic acid on angiogenesis in each step by using in vitro angiogenesis assays. We first tested whether pyruvic acid is cytotoxic in BAECs. Because pyruvic acid is a natural metabolite in mammalian cells, it is not surprising that pyruvic acid showed no cytotoxicity in BAECs. We also observed the stimulation of BAEC DNA synthesis by pyruvic acid (Fig. 3,A). The cells incubated in the absence of pyruvic acid, lose their proliferation activity in a time-dependent manner (data not shown). In addition, pyruvic acid strongly enhances the ability of BAECs migration in a dose-dependent manner, and this activity occurs in a short time, with just 4 h of pyruvic acid treatment (Fig. 3 B).
One of the aspects of the biological relevance of endothelial cell chemotaxis in vitro that is most important to the process of angiogenesis is the ability to promote morphological differentiation into capillary-like structure. In the presence of pyruvic acid, BAECs, placed on the growth factor-reduced Matrigel, formed short and thick capillary-like networks that seemed to be indicative of an early stage of angiogenic development, whereas BAECs in the absence of pyruvic acid established no tube-like structure (Fig. 3 C). By performing these in vitro angiogenesis assays, we concluded that pyruvic acid has strong angiogenic effect on these sequential angiogenic cascades.
In addition, tumors produce a wide array of angiogenic molecules during angiogenic process. So we checked the involvement of pyruvic acid on several major angiogenic factors and receptors activation in neovascularization. As shown in Fig. 4, the expression of VEGF and FGFR-2 were markedly stimulated by treatment of pyruvic acid for 4 h in BAECs. Therefore, we suggest the possibility that pyruvic acid stimulates BAECs angiogenic actions involving proliferation, migration, and differentiation of endothelial cells through secretion and auto-regulation of direct angiogenic factors and much more acceptation of angiogenic signals by angiogenic receptors.
It has been known that a potent angiogenic factor, VEGF, was up-regulated by the HIF-1 in cancer cells (19). It has also been reported that the HIF-1 activates transcription of genes that encode glycolytic enzymes in hypoxic cells (20). Therefore, the HIF-1 seems to play a central role in hypoxia-induced angiogenesis by the up-regulation of angiogenic factors such as VEGF and also by transcriptional activation of glycolytic enzymes, which leads to high production of pyruvic acid in cancer cells. Consequently, the pyruvic acid secreted by tumors triggers new blood vessel formation in endothelial cells by the over-expression of VEGF and FGFR-2.
Taken together, the observations in the present study suggest that pyruvic acid exhibits strong angiogenic actions and also may have the potential to be a useful activator of the large number of serious diseases characterized by deregulated angiogenesis. Further study is required to define more precisely the molecular mechanisms by which pyruvic acid modulates endothelial cell function and gene expression as well as the pathological relevance of these findings.
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Supported in part by the National Research Laboratory fund, the Ministry of Science and Technology, Korea (to K-W. K.), and Pusan National University Research Grant (to Y-J. K.).
The abbreviations used are: CAM, chorioallantoic membrane; BAEC, bovine aortic endothelial cell; FGFR-2, fibroblast growth factor receptor 2; VEGF, vascular endothelial growth factor; RT-PCR, reverse transcription-PCR; bFGF, basic FGF; HIF-1, hypoxia-inducible factor-1; PMA, phorbol 12-myristate 13-acetate.